JP2021523227A - Methods and Compositions for the Treatment of Cholesteryl Ester Accumulation - Google Patents

Abstract

Provided are methods and compositions for treating subjects in need of treatment, such as subjects with deficient LAL protein expression or subjects with cholesteryl ester accumulation disease.
[Selection diagram] Fig. 1

Description

Cross-reference of related applications
[0001] The present application claims the interests of US Provisional Application No. 62 / 667,205 filed May 4, 2018, which is incorporated herein by reference in its entirety.

[0002] Cholesteryl ester accumulation disease (CESD) (sometimes also referred to as lysosomal acid lipase (LAL) deficiency) is an autosomal recessive chronic liver disease caused by a deficiency of the functional LAL enzyme. CESD is a rare but serious illness that occurs when the body does not produce enough LAL. The LAL enzyme (also called cholesteryl ester hydrolase) is an enzyme that breaks down fatty substances and is essential for the metabolism of cholesteryl ester and triglyceride. Point mutations in the LIPA gene result in the production of short-chain proteins lacking enzymatic activity, leading to the accumulation of cholesteryl esters in organs and tissues. Reduced LAL enzyme activity typically results in massive deposition of fatty material in various tissues, including the liver, spleen, intestines, vascular walls, and other important organs. As a result, CESD is typically associated with significant morbidity and mortality and can affect individuals from infancy to adulthood.

[0003] According to one aspect of the present disclosure, a method of treating a subject in need of treatment for cholesteryl ester accumulation disease (CESD) by regulating the expression of the target protein or functional RNA by the subject's cells. The cell has a skippable exon-containing pre-mRNA (SEC pre-mRNA), which is a skippable exson, an intron adjacent to the skippable exson 5'splice site, and a skippable exon. Containing an intron flanking the 3'splice site of Exxon, the SEC premRNA encodes the target protein or functional RNA, the method of which targets the cells of interest for the SEC premRNA encoding the target protein or functional RNA. Contact with an antisense oligomer (ASO) that is complementary to the chemical moiety, whereby skippable exons are retained in the mRNA processed from the SEC pre-mRNA encoding the target protein or functional RNA, thereby the subject. Provided herein are methods comprising regulating the level of mRNA encoding a target protein or functional RNA and regulating the expression of the target protein or functional RNA in the cells of.

[0004] According to one aspect of the present disclosure, a method of regulating the expression of a target protein, which is a lithosome acid lipase (LAL), by cells having a skippable exon-containing premRNA (SEC premRNA), the SEC. PremRNAs include skippable exons, introns flanking the 5'splice site of skippable exons, and introns flanking the 3'splice site of skippable exons, where the SEC premRNA encodes the LAL protein. The method contacts cells with an antisense oligomer (ASO) that is complementary to the targeted portion of the SEC premRNA encoding the LAL protein, whereby skippable exons are SEC premRNA encoding the LAL protein. Provided herein are methods that include being retained in an mRNA processed from, thereby regulating the level of the mRNA encoding the LAL protein and thus the expression of the LAL protein in the cell.

[0005] In some embodiments, regulating the expression or level of the target protein or mRNA encoding the target protein increases the expression or level of the target protein or mRNA encoding the target protein. In some embodiments, regulating the expression or level of the LAL protein or mRNA encoding the LAL protein increases the expression or level of the LAL protein or mRNA encoding the LAL protein. In some embodiments, the target protein is LAL. In some embodiments, the cells are within or derived from a subject having a pathology caused by a deficiency in the amount or activity of the LAL protein. In some embodiments, at least one of the nine nucleotides at +1 to +6 of the intron adjacent to the 5'splice site and -3e to -1e of the skippable exon is at least one. Includes mutations. In some embodiments, the mutation is a substitution. In some embodiments, the mutation is at -1e. In some embodiments, the mutation is c. 894G> A. In some embodiments, the deficiency in the amount of target protein is caused by autosomal recessive inheritance. In some embodiments, the subject has a first allelic gene encoding a functional target protein and (a) a second allelic gene encoding a functional target protein, the antisense oligomer being the first or The antisense oligomer binds to the targeted portion of the SEC premRNA transcribed from the second allelic gene, or has said first allelic gene and (b) second allelic gene, and the antisense oligomer is the first allogeneic. It binds to the targeted portion of the SEC premRNA transcribed from the gene. In some embodiments, the subject has a pathology caused by a disorder caused by a deficiency in the amount or function of the target protein, and the subject is a first allelic gene comprising a first mutation, the first. Due to the mutation, the target protein is produced at a reduced level compared to the production from the wild-type allelic gene, or the target protein is produced in a form having reduced function compared to the equivalent wild-type protein. A second allelic gene, including a first allelic gene and a second mutation, in which the target protein is not produced, and the second mutation causes the target protein to be compared to the production from the wild-type allelic gene. Has a second allelic gene that is produced at reduced levels, the target protein is produced in a form that has reduced function compared to the equivalent wild-type protein, or the target protein is not produced. , 1st and 2nd mutations are the same or different. In some embodiments, the target protein is produced in a form with reduced function compared to an equivalent wild-type protein. In some embodiments, the target protein is produced in a fully functional form as compared to an equivalent wild-type protein. In some embodiments, the targeted portion of the SEC premRNA is from -4e to the skippable exon 3'splice site within the skippable exon + 2e to the skippable exon 3'splice site. Is in the area up to. In some embodiments, the targeted portion of the SEC premRNA is within the + 6 to +500 region of the intron adjacent to the 5'splice site of the skippable exon, or adjacent to the 3'splice site of the skippable exon. It is in the range of -16 to -500 of the intron. In some embodiments, the targeted portion of the SEC premRNA is within the region -4e to -500e for the 5'splice site of the skippable exon and +6 for the 5'splice site of the skippable exon. Within the region ~ + 500, within the region -16 to -500 with respect to the skippable exon 3'splice site, or within the region + 2e to + 500e with respect to the skippable exon 3'splice site. In some embodiments, the SEC premRNA has at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NO: 1 or its complementary sequences. Includes sequences with. In some embodiments, the SEC premRNA is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 1 or its complementary sequence. Will be done. In some embodiments, the targeted portion of the SEC premRNA is at least 80%, 85%, 90%, 95% in a region containing at least 8 contiguous nucleic acids of SEQ ID NOs: 2-4 or their complementary sequences. , 97%, or 100% sequence identity. In some embodiments, the ASO has at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 5-63 or their complementary sequences. Contains an array. In some embodiments, the targeted portion of the SEC premRNA is within the region from -4e to the skippable exon 5'splice site to + 2e to the skippable exon 3'splice site. , The exon that can be skipped is exon 8. In some embodiments, the ASO has at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 54-63 or their complementary sequences. Contains an array. In some embodiments, the targeted portion of the SEC premRNA is within the region +6 to +500 relative to the 5'splice site of the skippable exon, and the skippable exon is exon 8. In some embodiments, the ASO has at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 5-38 or their complementary sequences. Contains an array. In some embodiments, the targeted portion of the SEC premRNA is within the region -16 to -500 with respect to the 3'splice site of the skippable exon, and the skippable exon is exon 8. In some embodiments, the ASO has at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 39-53 or their complementary sequences. Contains an array. In some embodiments, the SEC pre-mRNA is either a partially spliced pre-mRNA of the full-length pre-mRNA or a partially spliced pre-mRNA of the wild-type pre-mRNA. In some embodiments, the mRNA encoding the target protein or functional RNA is full-length mature mRNA or wild-type mature mRNA. In some embodiments, the target protein produced is a full-length protein, a wild-type protein, or a combination thereof.

[0006] In some embodiments, the total amount of mRNA encoding the target protein or functional RNA produced intracellularly in contact with the antisense oligomer is the target protein or functional RNA produced intracellularly. About 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, compared to the total amount of mRNA encoding About 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 Double, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times , About 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold increase.

[0007] In some embodiments, the total amount of mRNA encoding the target protein or functional RNA produced intracellularly in contact with the antisense oligomer is the target protein or functional RNA produced intracellularly. About 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, as compared to the total amount of mRNA encoding , About 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% ~ About 250%, about 200% ~ about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least Increases by about 300%.

[0008] In some embodiments, the total amount of target protein produced by cells in contact with the antisense oligomer is about 1.1 to about 10 compared to the total amount of target protein produced by control cells. Double, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, About 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 ~ About 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to About 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least It increases by about 4 times, at least about 5 times, or at least about 10 times.

[0009] In some embodiments, the total amount of target protein produced by cells in contact with the antisense oligomer is about 20% to about 300% relative to the total amount of target protein produced by control cells. , About 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% ~ About 150%, about 100% ~ about 200%, about 100% ~ about 250%, about 150% ~ about 200%, about 150% ~ about 250%, about 200% ~ about 250%, at least about 10%, It increases by at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%.

[0010] In some embodiments, the antisense oligomer comprises a skeletal modification that includes a phosphorothioate ligation or a phosphorodiamidate ligation. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety.

[0011] In some embodiments, the antisense oligomer contains 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25. Nucleic acid bases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases Nucleic acid bases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases , 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 ~ 35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases, 12-50 nucleobases, 12-40 It consists of 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

[0012] In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% of the targeted portion of the SEC premRNA encoding the protein. , Or 100% complementarity. In some embodiments, the method further comprises assessing LIPA protein expression. In some embodiments, CESD is treated, the antisense oligomer binds to the targeting portion of the LIPA SEC premRNA, and the targeting portion is at least eight contiguous nucleic acids of SEQ ID NOs: 2-4 or their complementary sequences. including. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a foetation, embryo, or pediatric. In some embodiments, the cells are ex vivo contacted. In some embodiments, the antisense oligomer is administered by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection into the subject.

[0013] In some embodiments, the 16 nucleotides in the skippable exon + 1e adjacent to the -15--1 and 3'splice sites of the intron are identical to the corresponding wild-type sequence and the intron. Is an intron 7, and the skippable exon is an exon 8.

[0014] According to another aspect of the present disclosure, an antisense oligomer used herein is provided for any one of the methods provided herein.
[0015] According to another aspect of the present disclosure, at least about 80%, 85%, 90%, 95%, 97%, or 100% of the sequences in any one of SEQ ID NOs: 5-63 or their complementary sequences. Antisense oligomers containing sequences with identity are provided herein.

[0016] According to another aspect of the present disclosure, a pharmaceutical composition comprising an antisense oligomer and a pharmaceutically acceptable excipient, diluent, or carrier is provided herein.
Subjects in need of treatment by administering the pharmaceutical composition according to claim 52 by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. How to treat.

[0018] According to another aspect of the present disclosure, a cellular target protein or function for treating a cholesteryl ester accumulation disease (CESD) associated with a deficient protein or deficient functional RNA in a subject in need of treatment. A composition comprising an antisense oligomer for use in a method of regulating the expression of sex RNA, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, and the antisense oligomer is the target protein. Alternatively, it reduces the removal of exons from skippable exson-containing premRNA (SEC premRNA) that encodes functional RNA, targeting protein is deficient protein, functional RNA is deficient RNA, and SEC premRNA is The skippable exson encodes a target protein or functional RNA, including a skippable exson, an intron adjacent to the 5'splice site of the skippable exson, and an intron adjacent to the 3'splice site of the skippable exson. A composition is provided herein that is retained in a processed mRNA from a SEC pre-mRNA that regulates the production or activity of a target protein or functional RNA in a subject. A composition comprising an antisense oligomer for use in a method of treating a pathological condition associated with a lysosomal acid lipase (LAL) protein in a subject in need of treatment of the LAL protein by the subject cell. Including regulating expression, the cell contains a skippable exon containing a skippable exon, an intron adjacent to the 5'splice site of the skippable exon, an intron adjacent to the 3'splice site of the skippable exon. Having a pre-mRNA (SEC pre-mRNA), the SEC pre-mRNA encodes the LAL protein, the method of which contacts the cells with an antisense oligomer, whereby skippable exons encode the LAL protein. Also provided herein are compositions that are retained in processed mRNAs from mRNA transcripts, thereby regulating the level of mRNA encoding the LAL protein and thus regulating the expression of the LAL protein in cells of interest. ..

[0019] In some embodiments, regulating the activity, expression, and / or production of the target protein or mRNA encoding the target protein is the activity, expression, and / or regulation of the target protein or mRNA encoding the target protein. Or increase production. In some embodiments, regulating the activity, expression, and / or production of the LAL protein or mRNA encoding the LAL protein results in the activity, expression, and / or production of the LAL protein or mRNA encoding the LAL protein. increase. In some embodiments, the target protein is LAL. In some embodiments, the condition is a disease or disorder. In some embodiments, the disease or disorder is CESD. In some embodiments, the target protein and SEC premRNA are encoded by the LIPA gene. In some embodiments, the ASO is in the region from -4e for the 5'splice site of the skippable exon to + 2e for the 3'splice site of the skippable exon within the skippable exon. Target the portion of SEC pre-mRNA. In some embodiments, the antisense oligomer is in the region of +6 to +500 relative to the 5'splice site of the skippable exon, or in the 3'splice site of the skippable exon. In contrast, the portion of SEC premRNA within the region -16 to -500 is targeted. In some embodiments, the antisense oligomer is located within a region approximately 100 nucleotides downstream of the skippable exon 5'splice site to approximately 100 nucleotides upstream of the skippable exon 3'splice site. Target the part of. In some embodiments, the targeted portion of the SEC premRNA is within the + 6 to +500 region of the intron adjacent to the 5'splice site of the skippable exon, or adjacent to the 3'splice site of the skippable exon. It is in the range of -16 to -500 of exons. In some embodiments, the targeted portion of the SEC premRNA is within the region -4e to -500e for the 5'splice site of the skippable exon and +6 for the 5'splice site of the skippable exon. Within the region ~ + 500, within the region -16 to -500 with respect to the skippable exon 3'splice site, or within the region + 2e to + 500e with respect to the skippable exon 3'splice site. In some embodiments, the target protein is LAL. In some embodiments, the SEC premRNA has at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of SEQ ID NO: 1 or its complementary sequences. Includes sequences with. In some embodiments, the SEC premRNA is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 1 or its complementary sequence. Will be done. In some embodiments, the targeted portion of the SEC premRNA is at least 80%, 85%, 90%, 95% in a region containing at least 8 contiguous nucleic acids of SEQ ID NOs: 2-4 or their complementary sequences. , 97%, or 100% sequence identity. In some embodiments, the ASO has at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 5-63 or their complementary sequences. Contains an array. In some embodiments, the targeted portion of the SEC premRNA is within the region from -4e to the skippable exon 5'splice site to + 2e to the skippable exon 3'splice site. , The exon that can be skipped is exon 8. In some embodiments, the ASO has at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 54-63 or their complementary sequences. Contains an array. In some embodiments, the targeted portion of the SEC premRNA is within the region -16 to -500 with respect to the 3'splice site of the skippable exon, and the skippable exon is exon 8. In some embodiments, the ASO has at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 5-38 or their complementary sequences. Contains an array. In some embodiments, the targeted portion of the SEC premRNA is within the region +6 to +500 relative to the 5'splice site of the skippable exon, and the skippable exon is exon 8. In some embodiments, the ASO has at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 39-53 or their complementary sequences. Contains an array. In some embodiments, the SEC pre-mRNA is produced by partial splicing of full-length pre-mRNA or wild-type pre-mRNA. In some embodiments, the mRNA encoding the target protein or functional RNA is full-length mature mRNA or wild-type mature mRNA. In some embodiments, the target protein produced is a full-length protein or a wild-type protein. In some embodiments, the antisense oligomer comprises a skeletal modification that includes a phosphorothioate ligation or a phosphorodiamidate ligation. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomer contains 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases. Bases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 10 30 nucleobases, 10-25 nucleobases, 10-20 nucleobases, 10-15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 Nucleic Acid Bases, 11-30 Nucleic Acid Bases, 11-25 Nucleic Acid Bases, 11-20 Nucleic Acid Bases, 11-15 Nucleic Acid Bases, 12-50 Nucleic Acid Bases, 12-40 Nucleic Acids It consists of bases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases.

[0020] Also provided herein are pharmaceutical compositions comprising an antisense oligomer of any of the compositions according to claims 54-86 and a pharmaceutically acceptable excipient, diluent, or carrier. NS.
[0021] According to another aspect of the present disclosure, the pharmaceutical composition according to claim 87 is administered by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. Thereby provided herein is a method of treating a subject in need of treatment.

[0022] According to another aspect of the present disclosure, in a pharmaceutical composition comprising an antisense oligomer that hybridizes to the target sequence of the LIPA mRNA transcript and a pharmaceutically acceptable excipient, diluent, or carrier. Provided herein are pharmaceutical compositions in which the LIPA mRNA transcript comprises skipped exons and the antisense oligomer induces the retention of skipped exons in the LIPA mRNA transcript. In some embodiments, the LIPA mRNA transcript is a LIPA SEC pre-mRNA transcript. In some embodiments, the targeted portion of the LIPA SEC pre-mRNA transcript is from -4e to the skippable exon 3'splice site relative to the skippable exon 5'splice site within the skippable exon. On the other hand, it is in the area up to + 2e. In some embodiments, the LIPA SEC pre-mRNA transcript is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% in any one of SEQ ID NO: 1 or its complementary sequences. , 99%, or 100% sequence identity. In some embodiments, the LIPA SEC pre-mRNA transcript is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% in any one of SEQ ID NO: 1 or its complementary sequences. , 99%, or 100% sequence identity. In some embodiments, the antisense oligomer comprises a skeletal modification that includes a phosphorothioate ligation or a phosphorodiamidate ligation. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, the antisense oligomer contains 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases. Bases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 10 30 nucleobases, 10-25 nucleobases, 10-20 nucleobases, 10-15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 Nucleic Acid Bases, 11-30 Nucleic Acid Bases, 11-25 Nucleic Acid Bases, 11-20 Nucleic Acid Bases, 11-15 Nucleic Acid Bases, 12-50 Nucleic Acid Bases, 12-40 Nucleic Acids Includes nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases. In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% on the targeted portion of the LIPA SEC pre-mRNA transcript. There is complementarity of. In some embodiments, the targeted portion of the LIPA SEC pre-mRNA transcript is within a sequence selected from SEQ ID NOs: 2-4 or their complementary sequences.

[0023] In some embodiments, the antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93% in any one of SEQ ID NOs: 5-63 or their complementary sequences. , 94%, 95%, 96%, 97%, 98%, or 99% contains nucleotide sequences with sequence identity.

[0024] In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 5-63 or complementary sequences thereof.
[0025] In some embodiments, the pharmaceutical composition is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

[0026] According to yet another aspect of the present disclosure, it is a method of treating a subject having a pathological condition caused by a deficiency in the amount or activity of the LAL protein, and is any one of SEQ ID NOs: 5-63 or complementary sequences thereof. Each includes a nucleotide sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Methods are provided herein that include administering an antisense oligomer to a subject. In some embodiments, the subject is a human.
Incorporation
[0027] All publications, patents, and patent applications described herein are presented as if each individual publication, patent, or patent application was incorporated specifically and individually. As incorporated herein by reference.

[0028] FIG. 1 is a schematic representation of LIPA premRNA. LIPA mutations that result in exon 8 skipping and ultimately cholesteryl ester accumulation (CESD) c. 894G> A is depicted. Schematic representations of mutant splice mRNA transcripts produced by wild-type and LIPA mutations are also depicted. As shown, the CESD mutation produces about 95% of the mRNA transcript in a short-chain form that results in a non-functional LAL protein, with only 3 to 3 functional (full-length) mRNAs that result in a functional LAL protein. Only 5% occurs. [0029] FIG. 2 shows the c.I. FIG. 5 illustrates an example of polyacrylamide gel electrophoresis (PAGE) showing that exon 8 skipping was confirmed in fibroblasts of CESD patients with the 894G> A mutation. RT-PCR analysis using fibroblasts from subjects and CESD patients and using primers for exons 7 and 9 was performed by c. The existence of a band corresponding to the skipping of exon 8 caused by the 894G> A mutation was confirmed. Product identity was confirmed by sequencing. [0030] FIG. 3 depicts an antisense oligonucleotide (ASO) walk in the 5'splice site region of an exemplary LIPA intron 8. Performed on the 5'splice site region of the LIPA intron 8 targeting sequences downstream of the 5'splice site using 2'-O-methoxyethyl (2'-MOE) ASO with a phosphorothioate (PS) backbone. A graph display of the completed ASO walk is shown. The ASO was designed to cover this region by shifting 1 nucleotide at a time from the +6 position to the +11 position and then shifting 5 nucleotides at a time. FIG. 4A depicts the ASO walk of the 5'splice site region of LIPA intron 8 as assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Typical PAGE is c. In fibroblasts of CESD patients with the 894G> A mutation, untreated (unt), mock-treated (Mock1, Mock2, by electroporation), or as described in the examples herein and in FIG. Shown are SYBR-Safe stained RT-PCR products of LIPA treated electroporated with 2'-MOE ASO targeting the 5'splice site region of intron 8 at a concentration of 120 nM. Two products corresponding to exon 8 content (overall, upper band) and exon 8 skipping (lower band) were quantified. [0032] FIG. 4B is a graph in which the total length (containing 8 exons)% is plotted from the data of FIG. 4A. The black line indicates that there is no change in terms of unprocessed (Unt). [0033] FIG. 5A is a diagram illustrating an exemplary dose-dependent effect of selected ASOs on fibroblasts of CESD patients. c. In fibroblasts of CESD patients with 894G> A mutations, mock treatment (Mock, by electroporation) or 2'-of A-3334 (+9) or A-3347 (+66) targeting intron 8. Representative PAGEs showing SYBR-Safe stained RT-PCR products of LIPA, electroporated at concentrations of 30 nM, 60 nM, 120 nM, 240 nM, and 480 nM by MOE ASO are shown. Two products corresponding to exon 8 content (overall, upper band) and exon 8 skipping (lower band) were quantified. [0034] FIG. 5B is a graph in which the overall length (containing 8 exons)% is plotted from the data of FIG. 5A. The black line indicates that there is no change in mock. [0035] FIG. 5C is a diagram illustrating an exemplary dose-dependent effect of selected ASOs on fibroblasts of CESD patients. c. 240 nM, 720 nM by mock treatment (Mock) or 2'-MOE ASO of A-3334 (+9) or A-3347 (+66) targeting intron 8 in fibroblasts of CESD patients with 894G> A mutations. , And representative PAGEs showing SYBR-Safe stained RT-PCR products of LIPA treated by electroporation at a concentration of 2160 nM are shown. Two products corresponding to exon 8 content (overall, upper band) and exon 8 skipping (lower band) were quantified. [0036] FIG. 5D is a graph in which the total length (containing 8 exons)% is plotted from the data of FIG. 5C. The black line indicates that there is no change in mock. [0037] FIG. 6A is a diagram illustrating an exemplary dose-dependent effect of selected ASOs on fibroblasts of CESD patients. c. Free in 1 μM, 5 μM, and 20 μM concentrations by mock treatment (Mock) or 2'-MOE ASO of A-3334 (+9) targeting intron 8 in fibroblasts of CESD patients with 894G> A mutations. Representative PAGEs showing SYBR-Safe stained RT-PCR products of LIPA treated with free-uptake are shown. Two products corresponding to exon 8 content (overall, upper band) and exon 8 skipping (lower band) were quantified. [0038] FIG. 6B is a graph in which the total length (containing 8 exons)% is plotted from the data of FIG. 6A. The black line indicates that there is no change in mock. [0039] FIG. 7A is a diagram illustrating an exemplary dose-dependent effect of selected ASOs on fibroblasts of CESD patients. c. In fibroblasts of CESD patients with 894G> A mutations, 0.125 μM, 0.5 μM, and 0.125 μM, 0.5 μM, and 2'-MOE ASO of mock-treated cells (-) or A-3334 (+9) targeting Intron 8. Representative sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showing lysosomal acid lipase (LAL) protein from cells treated with electroporation at a concentration of 2 μM is shown. The product corresponding to the glycosylated full-length protein was quantified and normalized to a Ponso S-stained blot. [0040] FIG. 7B is a graph plotting the rate of change of the glycosylated full-length protein compared to mock from the data of FIG. 7A. [0041] FIG. 8 is electroporated at concentrations of 0.125 μM, 0.5 μM, and 2 μM by mock treatment (CESD control) or A-3334 (+9) 2'-MOE AOS targeting intron 8. Processed, c. 6 is a graph plotting the dynamics of LAL activity measured at 5 minute intervals from 0 to 60 minutes for protein extracts from fibroblasts of CESD patients with the 894G> A mutation. Protein extracts from wild-type fibroblasts (wild-type controls) are included for comparison. Relative fluorescence units (RFU) per μg of protein are plotted. [0042] FIG. 9A illustrates the exemplary dose-dependent effect of selected ASOs of different lengths targeting the +9 region of intron 8 of fibroblasts in CESD patients. c. In fibroblasts of CESD patients with the 894G> A mutation, mock treatment (control) or targeting intron 8 A-3334 (+9), A-3680 (+9 (17)), A-3681 (+9 (+9)) 16)), A-3682 (+10 (17)), A-3683 (+10 (16)), A-3684 (+111 (16)) by 2'-MOE ASO 0.125 μM, 0.5 μM, and 2 μM Representative PAGE showing SYBR-Safe stained RT-PCR products of LIPA, treated with electroporation at the concentration of. Two products corresponding to exon 8 content (overall, upper band) and exon 8 skipping (lower band) were quantified. [0043] FIG. 9B is a graph in which the total length (containing 8 exons)% is plotted from the data of FIG. 9A. [0044] FIG. 10 shows A-3334 (+9), A-3680 (+9 (17)), A-3681 (+9 (16)), A-3682, which are mocked (control) or target intron 8. Electroporation at concentrations of 0.125 μM, 0.5 μM, and 2 μM by 2'-MOE AOS of (+10 (17)), A-3683 (+10 (16)), A-3684 (+111 (16)). Processed, c. 6 is a graph plotting changes in LAL activity (curve slope) measured from 30 minutes to 60 minutes for protein extracts from fibroblasts of CESD patients with the 894G> A mutation. Changes in RFU per minute per 1 μg of protein are plotted.

Detailed description of the invention

[0045] Certain details of this description are provided to provide a good understanding of the various embodiments. However, one of ordinary skill in the art understands that the present disclosure may be implemented without these details. In other cases, well-known structures are not shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless required by context, the word "comprise" and its variants, such as "comprises" and "comprising", are open throughout the specification and subsequent claims. It should be interpreted as having a comprehensive meaning, that is, "including, but not limited to". Moreover, the headings provided herein are for convenience only and do not explain the scope or intent of the alleged disclosure.

[0046] As used in this specification and the appended claims, the articles "a", "an" and "the" include more than one subject unless explicitly stated in the content. It should also be noted that the term "or" is commonly used in the sense that "and / or" is included, unless explicitly stated in the content.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and substances similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, but suitable methods and substances are described below.

Cholesteryl ester accumulation disease and LIPA gene
[0048] Cholesteryl ester accumulation disease (CESD) is an autosomal recessive chronic liver disease caused by a functional lysosomal acid lipase (LALD) enzyme deficiency. The LAL enzyme is essential for the metabolism of cholesteryl ester and, to a lesser extent, also for the metabolism of triglycerides. Deficiency of LAL activity mainly results in the accumulation of cholesteryl ester in organs and tissues, especially the liver, spleen, and macrophages. The disease is characterized by hypercholesterolemia, hypertriglyceridemia, HDL deficiency, and abnormal lipid deposition in many organs. In the liver, hepatomegaly is caused by fatty liver and by fibrosis, which can lead to nodular cirrhosis. Life expectancy of patients is often less than 30 years of age. The onset of CESD typically occurs in childhood or adolescence, but symptoms may not be detected until adulthood and are almost equally prevalent in women and men.

[0049] Mutations in the LIPA gene encoding the LAL protein are the underlying cause of CESD. Unlike Wolman's disease, where a LIPA gene mutation produces an enzyme with no residual activity or no enzyme at all, a mutation that causes CESD encodes a LAL that retains some enzyme activity. Previous studies on LAL activity in cultured dermal fibroblasts of CESD patients have shown that a G at position -1 of the 5'splice site of exon 8 results in a 72 base pair (bp) inframe deletion from the resulting mRNA. → A mutation (E8SJM, exon 8 splice junction mutation, c.894G> A) was confirmed. This 72bp deletion was then shown to correspond to a full-length exon 8. The G → A mutation causes abnormal splicing of LIPA premRNA and abnormal skipping of exons 8. Deletion of exon 8 from mRNA results in codon loss of amino acids 254 to 277 of the LAL protein. The shortened protein has no residual LAL activity, but E8SJM does not cause Wolman's disease because 2% to 5% of normally spliced LIPA is present on the homozygous carrier. The majority of CESD patients described to date are E8SJM carriers.

[0050] The present disclosure is based on LIPA's c. Provided are compositions and methods that regulate the aberrant splicing caused by the 894G> A mutation to increase the production of mature mRNA encoding a functional protein and thus increase the translated functional LAL protein. These compositions and methods include antisense oligomers (ASOs) that can promote exon content and constitutive splicing of RNA premRNA. In various embodiments, the methods of the present disclosure can be used to increase functional LAL protein to treat pathological conditions caused by LAL protein deficiency. In some embodiments, the condition is CESD.

[0051] In some embodiments, the methods of the invention are used to increase functional LAL protein production in subjects in need of treatment of the condition. In some embodiments, the subject does not necessarily have to be deficient in LAL as compared to wild-type, but in any case has a condition in which an increase in LAL reduces the condition. In some embodiments, CESD is caused by autosomal recessive inheritance.

Splicing
[0052] Intervening sequences or introns are large, highly dynamic, called spliceosomes, that regulate complex interactions between primary transcripts, small nuclear RNA (snRNA), and multiple proteins. It is removed by the RNA protein complex. Spliceosomes are ordered and specially assembled on each intron to facilitate binding of U2 snRNA from U1 snRNA recognition of the 5'splice site (5'ss) or to bifurcation sequence (BPS). It starts with the recognition of 3'ss with binding of the cofactor (U2AF) to the 3'splice site (3'ss) region. U2AF is a stable heterodimer composed of a 65 kD subunit encoding U2AF2 (U2AF65) and a 35 kD subunit encoding U2AF1 (U2AF35), which binds to the polypyrimidine sequence (PPT) and U2AF35. Interacts with the highly conserved AG subunit at 3'ss to stabilize the binding of U2AF65. In addition to BPS / PPT units and 3'ss / 5'ss, for accurate splicing, an auxiliary sequence or structure that activates or suppresses splice site recognition, also known as an intron or exon splicing enhancer or silencer. is required. These elements allow the true splice site to be recognized in the genome of higher eukaryotes from a vast excess of potential or false sites that have the same sequence but are orders of magnitude higher than the authentic site. do. Although they often have regulatory functions, the exact mechanism of activation or inhibition is not well understood.

[0053] The determination of whether splicing occurs can typically be modeled as a stochastic process rather than a deterministic process, and even the most definitive splicing signals can sometimes be spliced inaccurately. However, under normal conditions, pre-mRNA splicing proceeds with surprisingly high accuracy. This may be partly due to the activity of adjacent cis-acting auxiliary exons and intron splicing regulatory sequences (ESRs or ISRs). Typically, these functional elements are classified as exon or intron splicing enhancers (ESE or ISE) or silencers (ESS or ISS), respectively, based on their ability to stimulate or inhibit splicing. There is currently evidence to suggest that some ancillary cis-acting elements may act by affecting the dynamics of spliceosome associations, eg, the placement of the complex between U1 snRNP and 5'ss. However, it seems likely that many elements will function in concert with trans-acting RNA-binding proteins (RBPs). For example, the serine and arginine-rich family of RBPs (SR proteins) is a conserved family of proteins that play a major role in determining exons. SR proteins promote exon recognition by recruiting components of the press spliceosome to adjacent splice sites or by antagonizing the effects of nearby ESS. The inhibitory effects of ESS and ISS are mediated by members of the heterogeneous ribonucleoprotein (hnRNP) family and can alter core splicing factor recruitment to adjacent splice sites. In addition to their role in splicing regulation, silencer elements have been suggested to play a role in suppressing pseudoexons, which have typical exon spacing but have a functional open reading frame. It is a set of no decoy intron splice parts. The ISE, ISS, ESE, and ESS work with the associated trans-acting RBP to provide key components of a set of splicing controls that identify when, where, and how mRNA is assembled from precursors. show.

The sequences that make up the exon-intron boundary are degenerate signals of varying intensities that can occur frequently within human genes. In a multi-exon gene, different pairs of splice sites can be linked together in many different combinations to create a diverse transcript array from a single gene. This is commonly referred to as selective pre-mRNA splicing. Most mRNA isoforms produced by alternative splicing are excreted from the nucleus and translated into functional polypeptides, whereas different mRNA isoforms from a single gene can have significant variations in translation efficiency. Those mRNA isoforms that have an immature stop codon (PCT) at least 50 bp upstream of the exon junction complex can be targeted for degradation by the nonsense-mediated decay (NMD) pathway. Mutations in traditional (BPS / PPT / 3'ss / 5'ss) and ancillary splicing motifs cause abnormal pricing such as exon skipping, or latent (or false) exon content, or splice site activation. , Can contribute significantly to human morbidity and mortality. Abnormal and alternative splicing patterns can be affected by native DNA variants in exons and introns.

[0055] ESEs are very common and can be present in most, if not all, exons, including constitutive exons. Some exons may have abundant ESEs and are therefore resistant to point mutations, but in certain cases (eg, exons 8 in LIPA), single point mutations provide significant ESEs. It can be destroyed, resulting in partially or completely inappropriate exon skipping.

Target transcript
[0056] In some embodiments, the methods of the present disclosure utilize the presence of skippable exon-containing pre-mRNA (SEC pre-mRNA) transcribed from the LIPA gene. Splicing of LIPA SEC pre-mRNA transcripts confirmed to produce functional mature LIPA mRNA can be induced using therapeutic agents such as ASO that promote exon-containing and constitutive splicing of LIPA SEC pre-mRNA. can. In some embodiments, the resulting functional mature LIPA mRNA is successfully translated, which can increase the amount of functional LAL protein in the patient's cells and alleviate the symptoms of CESD.

[0057] In various embodiments, the present disclosure provides therapeutic agents capable of targeting LIPA SEC pre-mRNA and regulating levels of splicing or protein expression. The therapeutic agent can be a small molecule, a polynucleotide, or a polypeptide. In some embodiments, the therapeutic agent is ASO. Various regions or sequences of LIPA SEC pre-mRNA can be targeted by therapeutic agents such as ASO. In some embodiments, the ASO targets the LIPA SEC premRNA transcribed from the LIPA gene. In some embodiments, the ASO targets the LIPA SEC premRNA transcribed from the LIPA gene, which contains skippable exons. In some embodiments, the skippable exon is exon 8 of the LIPA SEC pre-mRNA transcript. In some embodiments, skippable exons are excised from the LIPA SEC pre-mRNA transcript by aberrant splicing. In some embodiments, the aberrant splicing is caused by a mutation in the LIPA gene. In some embodiments, the mutation is a G → A mutation at position -1 of an exon 8 splice donor (E8SJM, exon 8 splice junction mutation, c.894G> A). In some embodiments, the ASO targets sequences within skippable exons of the LIPA SEC pre-mRNA transcript. In some embodiments, the skippable exon is exon 8 of the LIPA SEC pre-mRNA transcript. In some embodiments, the ASO targets the sequence within exon 8 of the LIPA SEC pre-mRNA transcript. In some embodiments, the ASO targets the exon sequence from the 5'splice site of exon 8 of the LIPA SEC premRNA transcript to the upstream (or 5'). In some embodiments, the ASO targets the exon sequence from the 3'splice site of exon 8 of the LIPA premRNA transcript to the downstream (or 3'). In some embodiments, the ASO targets sequences within the intron flanking the skippable exon 3'splice site of the LIPA SEC pre-mRNA transcript. In some embodiments, the ASO targets the sequence within the intron 7 of the LIPA SEC pre-mRNA transcript. In some embodiments, the ASO targets the intron sequence from the 3'splice site to the upstream (or 5') of the intron 7 of the LIPA SEC pre-mRNA transcript. In some embodiments, the ASO targets the intron sequence from the 5'splice site to the downstream (or 3') of the intron 7 of the LIPA premRNA transcript. In some embodiments, the ASO targets a sequence within the intron flanking the skippable exon 5'splice site of the LIPA SEC pre-mRNA transcript. In some embodiments, the ASO targets the sequence within Intron 8 of the LIPA SEC pre-mRNA transcript. In some embodiments, the ASO targets the intron sequence from the 3'splice site to the upstream (or 5') of the intron 8 of the LIPA SEC pre-mRNA transcript. In some embodiments, the ASO targets the intron sequence from the 5'splice site to the downstream (or 3') of the intron 8 of the LIPA premRNA transcript. In some embodiments, the ASO targets the sequence containing the exon-intron boundary of the LIPA SEC pre-mRNA transcript. In some embodiments, the exon is a skippable exon. The exon-intron boundary can represent the junction of exon and intron sequences. In some embodiments, the intron sequence can be flanked by the 5'end or 3'end of a skippable exon. In some embodiments, the ASO targets a sequence containing the exon 8-intron 8 boundary of the LIPA SEC pre-mRNA transcript. In some embodiments, the ASO targets a sequence containing the intro 7-exon 8 boundary of the LIPA SEC pre-mRNA transcript. In some embodiments, the ASO targets a sequence that contains both a portion of an intron and a portion of an exon.

[0058] In some embodiments, the ASO targets sequences from about 4 to about 300 nucleotides upstream (or 5') from the skippable exon 3'splice site of the LIPA SEC premRNA transcript. In some embodiments, the ASO is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 from the skippable exon 3'splice site of the LIPA SEC pre-mRNA transcript. Target sequences up to ~ about 150 nucleotides, about 150 ~ about 200 nucleotides, about 200 ~ about 250 nucleotides, or about 250 ~ about 300 nucleotides upstream (or 5'). In some embodiments, the ASO can target sequences from the skippable exon 3'splice site of the LIPA SEC pre-mRNA transcript to more than 300 nucleotides upstream. In some embodiments, the ASO targets sequences from about 4 to about 300 nucleotides downstream (or 3') from the skippable exon 3'splice site of the LIPA SEC premRNA transcript. In some embodiments, the ASO is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 from the skippable exon 3'splice site of the LIPA SEC pre-mRNA transcript. Target sequences ranging from ~ about 150 nucleotides, about 150 ~ about 200 nucleotides, about 200 ~ about 250 nucleotides, or about 250 ~ about 300 nucleotides downstream (or 3'). In some embodiments, the ASO can target sequences from the skippable exon 3'splice site of the LIPA SEC pre-mRNA transcript to more than 300 nucleotides downstream. In some embodiments, the ASO targets sequences from about 4 to about 300 nucleotides downstream (or 3') from the skippable exon 5'splice site of the LIPA SEC premRNA transcript. In some embodiments, the ASO is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 from the skippable exon 5'splice site of the LIPA SEC pre-mRNA transcript. Target sequences ranging from ~ about 150 nucleotides, about 150 ~ about 200 nucleotides, about 200 ~ about 250 nucleotides, or about 250 ~ about 300 nucleotides downstream. In some embodiments, the ASO targets sequences from the skippable exon 5'splice site of the LIPA SEC pre-mRNA transcript to more than 300 nucleotides downstream. In some embodiments, the ASO targets sequences from about 4 to about 300 nucleotides upstream (or 5') from the skippable exon 5'splice site of the LIPA SEC premRNA transcript. In some embodiments, the ASO is about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 from the skippable exon 5'splice site of the LIPA SEC pre-mRNA transcript. Target sequences up to ~ about 150 nucleotides, about 150 ~ about 200 nucleotides, about 200 ~ about 250 nucleotides, or about 250 ~ about 300 nucleotides upstream. In some embodiments, the ASO targets sequences from the skippable exon 5'splice site of the LIPA SEC pre-mRNA transcript to more than 300 nucleotides upstream.

When the LIPA gene (SEQ ID NO: 1) was analyzed for exon skipping events as described in the examples herein, removal of exon 8 from the processed mRNA from the SEC pre-mRNA was observed. rice field. In some embodiments, the ASOs disclosed herein target SEC premRNA transcribed from the LIPA genomic sequence. In some embodiments, the ASO targets SEC pre-mRNA transcripts from the LIPA genomic sequence containing skippable exons. In some embodiments, the skippable exon is exon 8. In some embodiments, the ASO targets SEC pre-mRNA transcripts from the LIPA genomic sequence containing exon 8. In some embodiments, the ASO is a SEC premRNA transcript from a LIPA genome sequence that comprises an intron flanking the 3'splice site of a skippable exon and an intron flanking the 5'splice site of a skippable exon. Target. In some embodiments, the intron adjacent to the 3'splice site of the skippable exon is the intron 7, and the intron adjacent to the 5'splice site of the skippable exon is the intron 8. In some embodiments, the ASO targets SEC pre-mRNA transcripts from the LIPA genomic sequence containing introns 7, exons 8, and introns 8. In some embodiments, the ASO targets the SEC pre-mRNA transcript of SEQ ID NO: 1. In some embodiments, the ASO targets the SEC pre-mRNA transcript of SEQ ID NO: 1 containing exon 8. In some embodiments, the ASO targets the SEC pre-mRNA transcript of SEQ ID NO: 1 containing intron 7. In some embodiments, the ASO targets the SEC pre-mRNA transcript of SEQ ID NO: 1 containing intron 8. In some embodiments, the ASO targets the SEC pre-mRNA transcript of SEQ ID NO: 1, which comprises intron 7, exon 8, and intron 8. In some embodiments, the ASO disclosed herein targets the LIPA SEC pre-mRNA sequence (SEQ ID NO: 1). In some embodiments, the ASO targets a LIPA SEC pre-mRNA sequence that includes skippable exons. In some embodiments, the ASO targets the LIPA SEC pre-mRNA sequence (SEQ ID NO: 4) containing exon 8. In some embodiments, the ASO targets a LIPA SEC pre-mRNA sequence containing an intron flanking the skippable exon 3'splice site. In some embodiments, the ASO targets the LIPA SEC pre-mRNA sequence (SEQ ID NO: 3), which comprises an intron 7. In some embodiments, the ASO targets a LIPA SEC pre-mRNA sequence containing an intron flanking the skippable exon 5'splice site. In some embodiments, the ASO targets the LIPA SEC pre-mRNA sequence (SEQ ID NO: 2) containing intron 8. In some embodiments, the ASO targets any one of the LIPA SEC pre-mRNA sequences of SEQ ID NOs: 2-4. In some embodiments, the ASO has any one of the sequences of SEQ ID NOs: 5-63.

[0060] In some embodiments, the LIPA SEC pre-mRNA transcript is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 to SEQ ID NO: 1 or its complementary sequence. It is encoded by a gene sequence having% or 100% sequence identity. In some embodiments, the LIPA SEC pre-mRNA transcript is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% in any one of SEQ ID NO: 1 or its complementary sequences. , 99%, or 100% sequence identity.

[0061] In some embodiments, the targeted portion of the LIPA SEC premRNA is at least 80%, 85%, 90 in a region containing at least 8 contiguous nucleic acids of SEQ ID NOs: 2-4 or their complementary sequences. Includes sequences with%, 95%, 97%, or 100% sequence identity. In some embodiments, the ASO has at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to any one of SEQ ID NOs: 5-65 or their complementary sequences. Contains an array.

[0062] In some embodiments, the ASO targets exon 8 of the LIPA SEC premRNA, which comprises skippable exons. In some embodiments, the ASO targets sequences from about 2 nucleotides downstream (or 3') of the 3'splice site of exon 8 to about 4 nucleotides upstream (or 5') of the 5'splice site of exon 8. To. In some embodiments, the ASO has a sequence of any one of SEQ ID NOs: 54-63 or their complementary sequences.

[0063] In some embodiments, the ASO targets the intron 7 of the LIPA SEC premRNA, which comprises skippable exons. In some embodiments, the ASO targets sequences from about 4 to about 300 nucleotides upstream (or 5') from the 3'splice site of intron 7. In some embodiments, the ASO targets sequences from about 16 to about 100 nucleotides upstream (or 5') from the 3'splice site of intron 7. In some embodiments, the ASO targets sequences from about 4 to about 300 nucleotides downstream (or 3') from the 5'splice site of intron 7. In some embodiments, the ASO has the sequence of any one of SEQ ID NOs: 39-53 or their complementary sequences.

[0064] In some embodiments, the ASO targets intron 8 of the LIPA SEC premRNA, which comprises skippable exons. In some embodiments, the ASO targets sequences from about 4 to about 300 nucleotides upstream (or 5') from the 3'splice site of intron 8. In some embodiments, the ASO targets sequences from about 4 to about 300 nucleotides downstream (or 3') from the 5'splice site of intron 8. In some embodiments, the ASO targets sequences 6 to about 100 nucleotides downstream (or 5') from the 5'splice site of intron 8. In some embodiments, the ASO has the sequence of any one of SEQ ID NOs: 5-38 or their complementary sequences.

[0065] In some embodiments, the targeted portion of the LIPA SEC pre-mRNA is an intron 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the targeted portion of the LIPA SEC premRNA is exons 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, hybridization of ASO to the targeted portion of SEC premRNA results in exon 8 inclusion, which in turn increases LAL protein production. In some embodiments, the targeted portion of the LIPA SEC premRNA is exon 8. In some embodiments, the targeted portion of the LIPA SEC premRNA is an intron 7. In some embodiments, the targeted portion of the LIPA SEC premRNA is an intron 8.

LAL protein
[0066] As also described above, the most common LIPA mutation in CESD occurs in exon 8 (eg, E8SJM, exon 8 splice junction mutation, c.894G> A). In some embodiments, c. The 894G> A mutation occurs in both alleles. In some embodiments, c. The 894G> A mutation occurs in one of two alleles. In some embodiments, the additional mutation occurs in one of the two alleles. In some embodiments, further mutations are described in c. It occurs in the same allele as the 894G> A mutation. In other embodiments, the further mutation is a trans mutation.

[0067] In some embodiments, the methods described herein are used to increase the production of functional LAL proteins. As used herein, the term "functionality" refers to the amount of activity or function of the LAL protein required to eliminate any one or more symptoms of the disease or condition being treated, eg, CESD. In some embodiments, the method is used to partially increase the production of functional LAL protein. As used herein, the term "partially functional" is more than any amount of activity or function required to eliminate or prevent any one or more symptoms of a disease or condition, eg, CESD. Represents any amount of activity or function of a low LAL protein. In some embodiments, the partially functional protein or RNA is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, as compared to the fully functional protein or RNA. It has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% less activity.

[0068] In some embodiments, the method is a method of increasing the expression of LAL protein by a subject cell having a SEC premRNA encoding the LAL protein, wherein the subject lacks the amount or activity of the LAL protein. Has CESD caused by, and a deficiency in the amount of LAL protein is caused by autosomal recessive inheritance. In such an embodiment, the subject is c. It has a first allele with a 984G> A mutation and a second allele from which the LAL protein is not produced. In another such embodiment, the subject is c. It has a first allele with a 984G> A mutation and a second allele encoding a non-functional LAL protein. In another such embodiment, the subject is c. It has a first allele with a 984G> A mutation and a second allele encoding a partially functional LAL protein. In another such embodiment, the subject is c. The first allele with the 984G> A mutation, and c. It has a second allele with a 984G> A mutation. In any of these embodiments, the antisense oligomer is c. It binds to the targeted portion of the SEC pre-mRNA transcribed from an allelic gene with the 984G> A mutation, thereby preventing skippable exon skipping from the pre-mRNA and producing the functional LAL protein in cells of interest. Increases the level of mature mRNA encoding and increases the expression of LAL protein.

[0069] In a related embodiment, the method is a method of using ASO to increase the expression of a functional protein or functional RNA. In some embodiments, ASO is used to increase expression of the LIPA protein in cells of interest having a SEC premRNA encoding the LAL protein, the subject having a deficiency in the amount or function of the LAL protein. For example, it has CESD.

[0070] In some embodiments, the SEC pre-mRNA transcript encoding the protein responsible for the disease or condition is targeted by the ASOs described herein. In some embodiments, the SEC pre-mRNA transcript, which encodes a protein that does not cause disease, is targeted by ASO. For example, diseases resulting from mutations or deficiencies of the first protein in a particular pathway are ameliorated by targeting NIEs containing pre-mRNA encoding the second protein, thereby a second protein. Production can be increased. In some embodiments, the function of the second protein can compensate for mutations or deficiencies (which are responsible for the disease or condition) of the first protein.

[0071] In some embodiments, the subject is
(A) LAL protein is produced at reduced levels compared to production from wild-type alleles, c. First mutant allele with 984G> A mutation, and (b)
(I) The LAL protein is c. Due to the 984G> A mutation, it is produced at reduced levels compared to production from wild-type alleles,
(Ii) LAL protein is produced at reduced levels compared to production from wild-type alleles,
(Iii) LAL protein is produced in a form with reduced function compared to equivalent wild-type protein, or (iv) LAL protein is not produced.
Second mutant allele,
Have,
SEC pre-mRNA can be found in c. It is transcribed from the first and / or second allele with the 984G> A mutation. In these embodiments, the ASO binds to the targeted portion of the SEC premRNA transcribed from the first or second allelic gene, thereby promoting exon content of A from the SEC pre-mRN. It causes an increase in the level of full-length mRNA encoding the LAL protein and an increase in the expression of the target protein or functional RNA in the cells of interest. In these embodiments, the target protein or functional RNA with increased expression levels as a result of exon content from the SEC premRNA is fully functional as compared to the equivalent wild-type protein (fully functional). ..

[0072] In some embodiments, the amount of LAL protein produced by contacting cells with ASO complementary to the targeted portion of the LIPA SEC pre-mRNA transcript is in the absence / treatment of ASO. At least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500 compared to the amount of protein produced by cells in the absence of Or increase by 1000%. In some embodiments, the total amount of LAL protein produced by cells contacted with the antisense oligomer is about 20% to about 300%, about 50, compared to the amount of target protein produced by the control compound. % ~ About 300%, about 100% ~ about 300%, about 150% ~ about 300%, about 20% ~ about 50%, about 20% ~ about 100%, about 20% ~ about 150%, about 20% ~ About 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150 %, About 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20 %, At least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%. In some embodiments, the total amount of LAL protein produced by cells contacted with the antisense oligomer is about 1.1 to about 10 times greater than the amount of target protein produced by the control compound. 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1. 1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 Double, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times , About 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times , At least about 5 times, or at least about 10 times. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the pre-mRNA.

[0073] In some embodiments, the level of mRNA encoding the LAL protein does not bind to, for example, cells not treated with antisense oligomers, or the targeted portion of LIPA SEC pre-mRNA in control cells. The amount is increased 1.1 to 10-fold compared to the amount of mRNA encoding the LAL protein produced in oligomer-treated cells.

[0074] In some embodiments, the level of mRNA encoding the LAL protein does not bind to, for example, cells untreated with antisense oligomers, or the targeted portion of LIPA SEC pre-mRNA in control cells. The amount is increased 1.1 to 10-fold compared to the amount of mRNA encoding the LAL protein produced in oligomer-treated cells.

[0075] In some embodiments of the invention, the subject may have a mutation in LIPA. Various pathogenic variants, including missense variants, nonsense variants, single and two base insertions and deletions, complex insertions / deletions, and splice site variants, have been reported to cause LAL deficiency. The most common pathogenic variant that produces CESD. 894G> a disrupts the normal donor splice consensus sequence with a G-to-A transition at -1e of exon 8 associated with the 5'splice site. Typically, this results in alternative splicing, followed by exon 8 skipping. In the presence of this pathogenic variant, approximately 2% to 5% of transcripts are accurately spliced, allowing residual enzyme activity. The catalytic active site of LAL is composed of amino acid residues Ser153, Asp324, and His353. The active site serine is part of the lipase consensus sequence that connects the β chain to the α-helix, known as the nucleophilic elbow, a nucleophile in a properly oriented complex. , Histidine, and ester carbon facilitate interaction. The disease results from loss of LAL function caused by a LIPA pathogenic variant that produces short-chain proteins or proteins with conformational changes or reduced activity.
[0076] In some embodiments, subjects known in the art and having any of the LIPA mutations described above can be treated using the methods and compositions described herein. .. In some embodiments, the mutation is in any LIPA intron or exon. In some embodiments, the mutation is in LIPA exon 8.

Exon skipping
[0077] As used herein, a "skipable exon-containing" or "SEC pre-mRNA" is a pre-mRNA transcript containing at least one skippable exon. Selective or aberrant splicing of SEC pre-mRNA can result in skipping of at least one skippable exon in the mature mRNA transcript. The terms "mature mRNA" and "fully spliced mRNA" are used interchangeably herein to describe fully processed mRNA. Skipping at least one skippable exon can result in unproductive mRNA. Mature mRNA can also result in abnormal protein expression.

The degree of exon skipping can be expressed as a percentage of exon skipping, eg, a percentage of the transcript in which a given exon is skipped. Briefly,% of exon skipping is calculated as a percentage of the amount of RNA transcripts skipped by exons to the sum of the average amount of RNA transcripts with exon skipping and the average amount of RNA transcripts containing exons. be able to.

[0079] In some embodiments, the skipped exons are at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30% from the RNA transcript. , An exon identified as a skipped exon, based on a removal decision of at least about 35%, at least about 40%, at least about 45%, or at least about 50%. In some embodiments, skipped exons are at least about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85% from RNA transcripts. , About 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% ~ About 20%, about 5% ~ about 15%, about 10% ~ about 100%, about 10% ~ about 95%, about 10% ~ about 90%, about 10% ~ about 85%, about 10% ~ about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50% , About 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 100%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% ~ About 70%, about 15% ~ about 65%, about 15% ~ about 60%, about 15% ~ about 55%, about 15% ~ about 50%, about 15% ~ about 45%, about 15% ~ about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90% , About 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 25% ~ About 100%, about 25% ~ about 95%, about 25% ~ about 90%, about 25% ~ about 85%, about 25% ~ about 80%, about 25% ~ about 75%, about 25% ~ about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40% , Or an exon identified as a skipped exon based on a removal decision of about 25% to about 35%. See ENCODE data (eg Tilgner, et al., 2012, “Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs,” Genome Research 22 (9): 1616-25. ) Can be used to assist in confirming exon skipping.

[0080] In some embodiments, the cells are produced by the cells in the absence of ASO / treatment by contacting the cells with an ASO that is complementary to the targeted portion of the LIPA premRNA transcript. Compared to the amount of protein, the amount of LAL protein produced is at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or Increase by 1000%. In some embodiments, the total amount of LAL protein produced by cells in contact with the antisense oligomer is about 20% to about 300%, about 50, compared to the amount of target protein produced by the control compound. % ~ About 300%, about 100% ~ about 300%, about 150% ~ about 300%, about 20% ~ about 50%, about 20% ~ about 100%, about 20% ~ about 150%, about 20% ~ About 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150 %, About 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20 %, At least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%. In some embodiments, the total amount of LAL protein produced by cells in contact with the antisense oligomer is about 1.1 to about 10 times greater than the amount of target protein produced by the control compound. 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1. 1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 Double, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times , About 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times , At least about 5 times, or at least about 10 times. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the pre-mRNA.

[0081] In some embodiments, the amount of LAL-encoding mRNA, including the mature mRNA encoding the target protein, by contacting the cell with an ASO complementary to the targeted portion of the LIPA pre-mRNA transcript. Will increase. In some embodiments, the amount of mRNA encoding the LAL protein or mature mRNA encoding the LAL protein is compared to the amount of protein produced by the cell in the absence of ASO / in the absence of treatment. Increase by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%. In some embodiments, the total amount of LAL protein-encoding mRNA or LAL protein-encoding mature mRNA produced intracellularly in contact with the antisense oligomer is in untreated cells, eg, untreated cells or controls. About 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300% compared to the amount of mature RNA produced in cells treated with the compound. , About 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% ~ About 200%, about 150% ~ about 250%, about 200% ~ about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200% , At least about 250%, or at least about 300% increase. In some embodiments, the total amount of LAL protein-encoding mRNA or LAL protein-encoding mature mRNA produced intracellularly in contact with the antisense oligomer is in untreated cells, eg, untreated cells or controls. About 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times the amount of mature RNA produced in cells treated with the compound. , About 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 Double, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times , At least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times. The control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the LIPA SEC premRNA.

Therapeutic agent
[0082] In various embodiments of the present disclosure, compositions and methods comprising therapeutic agents are provided to regulate protein expression levels of LAL. In some embodiments, compositions and methods for regulating alternative splicing of LIPA premRNA are provided herein. In some embodiments, compositions and methods for preventing exon skipping in splicing of LIPA premRNA, eg, preventing exon skipping in splicing of LIPA premRNA, are provided herein.

[0083] The therapeutic agent disclosed herein can be an exon skipping inhibitor. In some embodiments, the therapeutic agent may comprise a polynucleic acid polymer.
[0084] According to one aspect of the present disclosure, a method of treating or preventing a condition associated with a functional LAL protein deficiency is provided herein in which an exon skipping inhibitor is administered to a subject. Including increasing the level of functional LAL protein, the drug binds to certain regions of the pre-mRNA transcript and reduces exon skipping in the mature transcript. For example, a method for treating or preventing a condition associated with a functional LAL protein deficiency is provided herein in which an exon skipping inhibitor is administered to a subject to increase the level of the functional LAL protein. This agent binds to a region of the exon or intron of the premRNA transcript (eg, exon 8, intron 7, or intron 8 of the human LIPA gene).

[0085] When referring to the reduction of exon skipping in mature mRNA, the reduction may be complete, eg 100% or partial. The reduction can be clinically significant. The reduction / correction can be compared to the level of exon skipping in untreated subjects, or to the amount of exon skipping in a similar population of subjects. The reduction / correction can be at least 10% less exon skipping compared to the average or pretreatment subject. The reduction can be at least 20% less exon skipping compared to the average or pretreatment subject. The reduction can be at least 40% less exon skipping compared to the average or pretreatment subject. The reduction can be at least 50% less exon skipping compared to the average or pretreatment subject. The reduction can be at least 60% less exon skipping compared to the average or pretreatment subject. The reduction can be at least 80% less exon skipping compared to the average or pretreatment subject. The reduction can be at least 90% less exon skipping compared to the average or pretreatment subject.

[0086] When referring to an increase in functional LAL protein, the increase can be clinically significant. The increase can be compared to the level of functional LAL protein in untreated subjects, or to the amount of functional LAL protein in a similar population of subjects. The increase can be at least 10% more functional LAL protein compared to the average or pretreatment subject. The increase can be at least 20% more functional LAL protein compared to the average or pretreatment subject. The increase can be at least 40% more functional LAL protein compared to the average or pretreatment subject. The increase can be at least 50% more functional LAL protein compared to the average or pretreatment subject. The increase can be at least 80% more functional LAL protein compared to the average or pretreatment subject. The increase can be at least 100% more functional LAL protein compared to the average or pretreatment subject. The increase can be at least 200% more functional LAL protein compared to the average or pretreatment subject. The increase can be at least 500% more functional LAL protein compared to the average or pretreatment subject.

[0087] In embodiments where the exon skipping inhibitor comprises a polynucleic acid polymer, the length of the polynucleic acid polymer can be about 50 nucleotides. The length of the polynucleic acid polymer can be about 45 nucleotides. The length of the polynucleic acid polymer can be about 40 nucleotides. The length of the polynucleic acid polymer can be about 35 nucleotides. The length of the polynucleic acid polymer can be about 30 nucleotides. The length of the polynucleic acid polymer can be about 24 nucleotides. The length of the polynucleic acid polymer can be about 25 nucleotides. The length of the polynucleic acid polymer can be about 20 nucleotides. The length of the polynucleic acid polymer can be about 19 nucleotides. The length of the polynucleic acid polymer can be about 18 nucleotides. The length of the polynucleic acid polymer can be about 17 nucleotides. The length of the polynucleic acid polymer can be about 16 nucleotides. The length of the polynucleic acid polymer can be about 15 nucleotides. The length of the polynucleic acid polymer can be about 14 nucleotides. The length of the polynucleic acid polymer can be about 13 nucleotides. The length of the polynucleic acid polymer can be about 12 nucleotides. The length of the polynucleic acid polymer can be about 11 nucleotides. The length of the polynucleic acid polymer can be about 10 nucleotides. The length of the polynucleic acid polymer can be about 10 to about 50 nucleotides. The length of the polynucleic acid polymer can be about 10 to about 45 nucleotides. The length of the polynucleic acid polymer can be about 10 to about 40 nucleotides. The length of the polynucleic acid polymer can be about 10 to about 35 nucleotides. The length of the polynucleic acid polymer can be about 10 to about 30 nucleotides. The length of the polynucleic acid polymer can be about 10 to about 25 nucleotides. The length of the polynucleic acid polymer can be about 10 to about 20 nucleotides. The length of the polynucleic acid polymer can be from about 15 to about 25 nucleotides. The length of the polynucleic acid polymer can be from about 15 to about 30 nucleotides. The length of the polynucleic acid polymer can be from about 12 to about 30 nucleotides.

[0088] The sequence of the polynucleic acid polymer is at least 50%, 55%, 60%, 65%, 70%, 75%, 80% to the target sequence of the mRNA transcript, eg, the partially processed mRNA transcript. , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementarity. The sequence of the polynucleic acid polymer can be 100% complementary to the target sequence of the pre-mRNA transcript.

[0089] The sequence of the polynucleic acid polymer can have up to 4 mismatches with respect to the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer can have up to 3 mismatches with the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer can have no more than two mismatches with the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer can have one or less mismatches with the target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may not have a mismatch with the target sequence of the pre-mRNA transcript.

[0090] In some embodiments, the polynucleic acid polymer is capable of specifically hybridizing to the target sequence of the pre-mRNA transcript. For example, polynucleic acid polymers are 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% on the target sequence of the pre-mRNA transcript. Can have sequence complementarity. Hybridization can be under highly stringent hybridization conditions.

[0091] The polynucleic acid polymer is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% in the sequence selected from the group consisting of SEQ ID NOs: 5-63. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity. The polynucleic acid polymer can have a sequence of 100% sequence identity in the sequence selected from the group consisting of SEQ ID NOs: 5-63.

[0092] When referring to a polynucleic acid polymer sequence, one of ordinary skill in the art will maintain the ability to hybridize to the target sequence or be recognized as the target sequence if the substitution is within the target sequence. Understand that substitutions of are acceptable in the sequence, and in some cases two substitutions are acceptable. References to sequence identity can be determined by BLAST sequence alignment using standard / default parameters. For example, the sequences have 99% identity and can still have the functionality according to the present disclosure. In other embodiments, the sequences have 98% identity and can still have the functionality according to the present disclosure. In another embodiment, the sequences have 95% identity and can still have the functionality according to the present disclosure. In another embodiment, the sequences have 90% identity and can still have the functionality according to the present disclosure.

Antisense oligomer
[0093] Provided herein are compositions comprising antisense oligomers that prevent exon skipping by binding to targeted moieties of LIPA SEC pre-mRNA. As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and by Watson-Crick base pairing or wobble base pairing (GU) to target nucleic acids (eg, LIPA). Represents an oligomer such as a polynucleotide containing a nucleobase that hybridizes to a SEC pre-mRNA) sequence. The ASO can have accurate sequence complementarity or near complementarity to the target sequence (eg, sufficient complementarity to bind to the target sequence and enhance splicing at the splice site). The ASO is designed to bind (hybridize) to the target nucleic acid (eg, the targeted portion of the pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, when hybridizing to a site other than the target (targeted) nucleic acid sequence, it hybridizes to a limited number of sequences (several sites other than the target nucleic acid) that are not the target nucleic acid. The design of the ASO is such that the nucleic acid sequence of the target portion of the pre-mRNA transcript, or the genomic or intracellular pre-mRNA, is such that the ASO can bind to other sites and cause "off-target" effects. The generation of sufficiently similar nucleic acid sequences at other locations in the transcriptome can be taken into account. In the art, for example, any antisense oligomer known in the PCT application published as WO2015 / 03591, PCT / US2014 / 0545151, entitled "Reducing Nonsense-Mediated mRNA Decay," incorporated herein. Can be used to implement the methods described herein.

[0094] In some embodiments, the ASO is "specifically hybridizes" or "specifically" to the targeting portion of the target nucleic acid or RIC premRNA. _{Typically, such hybridization occurs at T m} , which is substantially higher than 37 ° C., preferably at least 50 ° C., typically 60 ° C. to approximately 90 ° C. Such hybridization preferably corresponds to stringent hybridization conditions. T _m is the temperature at which 50% of the target sequence hybridizes to the complementary oligonucleotide at a given ionic strength and pH.

[0095] Oligoomers, such as oligonucleotides, are "complementary" to each other when hybridization occurs in the antiparallel configuration between two single-stranded polynucleotides. A double-stranded polynucleotide can be "complementary" to another polynucleotide if hybridization can occur between one strand of the first polynucleotide and one strand of the second polynucleotide. .. Complementarity (to some extent that one polynucleotide is complementary to another) is that of bases in chains that are in different orientations and are predicted to form hydrogen bonds with each other according to generally accepted base pairing rules. It can be quantified in terms of proportions (eg, percentages). The sequence of the antisense oligomer (ASO) does not need to be 100% complementary to the sequence of the target nucleic acid in order to hybridize. In certain embodiments, the ASO is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% of the target region in the target nucleic acid sequence to be targeted. , At least 97%, at least 98%, or at least 99% sequence complementarity can be included. For example, an ASO in which 18 of the 20 nucleobases of an oligomeric compound are complementary to the target region and therefore specifically hybridize represent 90% complementarity. In this example, the remaining non-complementary nucleobases may be clustered together or interspersed with complementary nucleobases, either contiguous with each other or in close proximity to complementary nucleobases. No. The% complementarity of ASO with the region of the target nucleic acid is the BLAST program (basic local alignment search tools) and the Power BLAST program known in the art (Altschul, et al., J. Mol. Biol., 1990, 215, 403). -410; Zhang and Madden, Genome Res., 1997, 7, 649-656) can be used to make routine decisions.

[0096] ASO does not have to hybridize with all nucleobases in the target sequence, and the nucleobases to hybridize may or may not be contiguous. The ASO can hybridize to one or more segments of the pre-mRNA transcript (eg, loop or hairpin structures can be formed) so that no intervening or adjacent segments are involved in the hybridization event. In certain embodiments, the ASO hybridizes to the discontinuous nucleobase of the target pre-mRNA transcript. For example, ASO can hybridize to nucleobases in pre-mRNA transcripts separated by one or more nucleobases to which ASO does not hybridize.

[0097] The ASOs described herein contain nucleobases that are complementary to the nucleobases present at the target portion of the SEC premRNA. The term ASO includes oligonucleotides and any other oligomeric molecules that include complementary nucleobases on the target mRNA but are free of sugar moieties such as peptide nucleic acids (PNAs). do. ASO can include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three thereof. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides having a modified or substituted glycosyl group and / or having a modified backbone. In some embodiments, all nucleotides of ASO are modified nucleotides. Chemical modifications of ASO or components of ASO that are compatible with the methods and compositions described herein are apparent to those of skill in the art and are hereby incorporated by reference in their entirety, U.S. Pat. No. 8,258. , 109B2, US Pat. No. 5,656,612, US Patent Application Publication No. 2012/0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 347-355.

[0098] One or more nucleobases of ASO are capable of hydrogen binding to any naturally occurring unmodified nucleobase such as adenin, guanine, cytosine, timine, and uracil, or nucleobases present in the target premRNA. It can be any synthetic or modified nucleobase that is sufficiently similar to the unmodified nucleobase. Examples of modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0099] The ASO described herein also includes a skeletal structure connecting the constituents of the oligomer. The terms "skeleton structure" and "oligomer linkage" can be used interchangeably and represent connections between monomers of ASO. In naturally occurring oligonucleotides, the backbone contains a 3'-5'phosphodiester bond that connects the sugar moieties of the oligomer. The skeletal structures or oligomeric linkages of ASOs described herein include, but are not limited to, phosphorothioates, phosphorodithioates, phosphoroserenoates, phosphorodiselenoates, phosphoroanilothioates. , Phosphoraniladate, phosphoramidate, etc. may be included. For example, LaPlanche, et al., Nucleic Acids Res. 14: 9081 (1986); Stec, et al., J. Am. Chem. Soc. 106: 6077 (1984); Stein, et al., Nucleic Acids Res. 16: 3209 (1988); Zon, et al., Anti Cancer Drug Design 6: 539 (1991); Zon, et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed. , Oxford University Press, Oxford England (1991)); Stec, et al., US Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90: 543 (1990). In some embodiments, the skeletal structure of ASO is phosphorus-free, rather, peptide bonds in, for example, peptide nucleic acids (PNAs), or linkages that include carbamate, amide, and linear and cyclic hydrocarbon groups. Contains groups. In some embodiments, the skeletal modification is a phosphothioate link. In some embodiments, the skeletal modification is a phosphoramidate link.

[0100] In some embodiments, each stereochemistry of the internucleotide linkage of phosphorus in the ASO backbone is random. In some embodiments, the respective stereochemistry of the internucleotide linkage of phosphorus in the ASO backbone is controlled and non-random. For example, US Patent Application Publication No. 2014/01/94610, "Methods for the Synthesis of Functionalized Nucleic Acids," incorporated herein, independently selects the chirality of each phosphorus atom in a nucleic acid oligomer. Describe the method. In some embodiments, the ASO used in the methods of the invention, including, but not limited to, any of the ASOs listed in Table 3, is an AOS having a non-random phosphorus internucleotide linkage. include. In some embodiments, the composition used in the method of the invention comprises pure diastereomeric ASO. In some embodiments, the compositions used in the methods of the invention are at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least. About 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% ~ About 100%, about 94% ~ about 100%, about 95% ~ about 100%, about 96% ~ about 100%, about 97% ~ about 100%, about 98% ~ about 100%, or about 99% ~ Contains ASO with about 100% diastereomeric purity.

[0101] In some embodiments, the ASO has a non-random mixture of Rp and Sp configurations in the internucleotide linkage of phosphorus. For example, it has been suggested that a mix of Rp and Sp in antisense oligomers is required to achieve a good balance between activity and nuclease stability (Wan, et al., 2014, “Synthesis, biophysical). properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages, "Nucleic Acids Research 42 (22): 13456-13468, incorporated herein by reference). In some embodiments, the ASO used in the methods of the invention, including, but not limited to, any of the ASOs listed in Table 3, is about 5-100% Rp, at least about 5%. Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp , At least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, the rest being Sp or containing about 100% Rp. In some embodiments, ASOs used in the methods of the invention, including, but not limited to, any of the ASOs listed in Table 3, have an Rp of about 10% to about 100%, about 15. % To about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, about 40 % To about 100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65 % To about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90 % To about 100% Rp, about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40 It contains% to about 60% Rp, or about 45% to about 55% Rp, the rest being Sp.

[0102] In certain embodiments, the ASO used in the methods of the invention, including, but not limited to, any of the ASOs listed in Table 3 is about 5-100% Sp, at least about 5. % Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% It contains Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, and the rest is Rp or contains about 100% Sp. In some embodiments, the ASO used in the method of the invention, including, but not limited to, any of the ASOs listed in Table 3, is about 10% to about 100% Sp. About 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, About 40% to about 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, About 65% to about 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, About 90% to about 100% Sp, about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, It contains about 40% to about 60% Sp, or about 45% to about 55% Sp, the rest being Rp.

[0103] Any ASO described herein contains a sugar moiety containing ribose or deoxyribose as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog containing a morpholine ring. can do. Non-limiting examples of the modified sugar moiety include 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F, N3'→ P5'phosphoroamidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanididium, 2'-O-guanidinium ethyl, carbamate-modified sugar, bicyclic modified sugar, etc. 2'substitution of. In some embodiments, the modification of the sugar moiety is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the modification of the sugar moiety is an extra cross-linking, such as in locked nucleic acids (LNAs). In some embodiments, the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification. In some embodiments, the sugar moiety comprises a 2'4'constrained 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety comprises a cEt 2', 4'restraint 2'-O-ethyl BNA modification. In some embodiments, the sugar moiety comprises a tricycloDNA (tDNA) modification. In some embodiments, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety comprises an MCE modification. Modifications are known in the art and are incorporated herein by reference, eg, Jarver, et al., 2014, “A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications,” Nucleic Acid Therapeutics. 24 (1): It is described in 37-47.

[0104] In some embodiments, each monomer of ASO is similarly modified, eg, each linkage of the backbone of ASO comprises a phosphorothioate linkage, or each ribose sugar moiety comprises a 2'O-methyl modification. Such modifications present in each of the monomer constituents of ASO are referred to as "uniform modifications". In some embodiments, a combination of different modifications may be desirable, for example, the ASO may include a combination of a phosphorodiamidate link and a sugar moiety containing a morpholino ring (morpholino). The combination of different modifications in ASO is referred to as "mixed modification" or "mixed chemistry".

[0105] In some embodiments, the ASO comprises one or more skeletal modifications. In some embodiments, the ASO comprises one or more partial sugar modifications. In some embodiments, the ASO comprises one or more skeletal modifications and one or more partial sugar modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs described herein, or any component of the ASO (eg, nucleobases, sugar moieties, scaffolds), is used to achieve the desired properties or activities of the ASO, or the undesired properties of the ASO. Can be modified to reduce activity. In some embodiments, the ASO or one or more components of any ASO is to enhance binding affinity for the target sequence on the pre-mRNA transcript; to reduce binding to any non-target sequence. To reduce degradation by intracellular nucleases (ie, RNase H); to improve ASO's approach to cells and / or cell nuclei: to alter the pharmacokinetics or pharmacodynamics of ASO, and to reduce the half-life of ASO. Can be modified to regulate.

[0106] In some embodiments, the ASO is composed of 2'-O- (2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOs composed of such nucleotides are particularly suitable for the methods described herein, and oligomers with such modifications have significantly enhanced resistance to nuclease degradation and increased bioavailability. For example, some embodiments described herein are suitable for oral delivery. See, for example, Geary, et al., J Pharmacol Exp Ther. 2001; 296 (3): 890-7; Geary, et al., J Pharmacol Exp Ther. 2001; 296 (3): 898-904.

Methods of synthesizing ASO are known to those of skill in the art. Alternatively or additionally, ASO can be obtained from commercial sources.
[0108] Unless otherwise specified, the left end of a single-stranded nucleic acid (eg, pre-mRNA transcript, oligonucleotide, ASO, etc.) sequence is the 5'end, leftward of the single-stranded or double-stranded nucleic acid sequence. Is referred to as the 5'direction. Similarly, the right end or right direction of a nucleic acid sequence (single or double strand) is the 3'end or 3'direction. Generally, a region or sequence 5'with respect to a nucleic acid origin is referred to as "upstream" and a region or sequence 3'with respect to a nucleic acid origin is referred to as "downstream". Generally, the start codon is located at the 5'or 5'end of the mRNA, and the stop codon is located at the 3'end or 3'end. In some embodiments, nucleotides upstream of the origin of the nucleic acid are designated as negative numbers, while nucleotides downstream of the origin are designated as positive numbers. For example, a base point (eg, an exon-exon junction in mRNA) is designated as a "zero" site, and a nucleotide directly adjacent to the base point and upstream is designated as "minus 1", eg, "-1", while , "Plus 1", for example "+1", is specified for nucleotides directly adjacent to the origin and downstream.

[0109] In some embodiments, the ASO is specified downstream (3'direction) of the skippable exon 5'splice site of the LIPA SEC premRNA (eg, a positive number for the 5'splice site). It is complementary (and binds) to the targeted portion of the LIPA SEC pre-mRNA at (direction). In some embodiments, the ASO is complementary to the targeted portion of the LIPA SEC premRNA that is within a region of about +6 to about +500 relative to the skippable exon 5'splice site. In some embodiments, the ASO is not complementary to +1 to +5 nucleotides relative to the 5'splice site (the first 5 nucleotides located downstream of the 5'splice site). In some embodiments, the ASO may be complementary to the targeted portion of the LIPA SEC premRNA that is within the region +6 to +100 with respect to the skippable exon 5'splice site. In some embodiments, the ASO is about +6 to about +500, about +6 to about +490, about +6 to about +480, about +6 to about +470, about +6 to the skippable Exxon 5'splice site. About +460, about +6 to about +450, about +6 to about +440, about +6 to about +430, about +6 to about +420, about +6 to about +410, about +6 to about +400, about +6 to about +390, about +6 to about +380 , About +6 to about +370, about +6 to about +360, about +6 to about +350, about +6 to about +340, about +6 to about +330, about +6 to about +320, about +6 to about +310, about +6 to about +300, about +6 to about +290, about +6 to about +280, about +6 to about +270, about +6 to about +260, about +6 to about +250, about +6 to about +240, about +6 to about +230, about +6 to about +220, about +6 to About +210, about +6 to about +200, about +6 to about +190, about +6 to about +180, about +6 to about +170, about +6 to about +160, about +6 to about +150, about +6 to about +140, about +6 to about +130 , About +6 to about +120, about +6 to about +110, about +6 to about +100, about +6 to about +90, about +6 to about +80, about +6 to about +70, about +6 to about +60, about +6 to about +50, about It is complementary to the targeted portion within the region of +6 to about +40, about +6 to about +30, or about +6 to about +20.

[0110] In some embodiments, the ASO is upstream (5'direction) of the skippable exon 3'splice site of the LIPA SEC premRNA (eg, a direction in which a negative number is specified for the 3'splice site). ) Is complementary (and binds) to the targeted portion of the LIPA SEC pre-mRNA. In some embodiments, the ASO is complementary to the targeted portion of the LIPA SEC premRNA that is within a region of about -16 to about -500 relative to the skippable exon 3'splice site. In some embodiments, the ASO is not complementary to -1 to -15 nucleotides relative to the 5'splice site (the first 5 nucleotides located downstream of the 5'splice site). In some embodiments, the ASO may be complementary to the targeted portion of the LIPA SEC premRNA that is within a region of about -16-100 nucleotides relative to the skippable exon 3'splice site. In some embodiments, the ASO is about +6 to about +500, about +6 to about +490, about +6 to about +480, about +6 to about +470, about +6 to the skippable Exxon 3'splice site. About +460, about +6 to about +450, about +6 to about +440, about +6 to about +430, about +6 to about +420, about +6 to about +410, about +6 to about +400, about +6 to about +390, about +6 to about +380 , About +6 to about +370, about +6 to about +360, about +6 to about +350, about +6 to about +340, about +6 to about +330, about +6 to about +320, about +6 to about +310, about +6 to about +300, about +6 to about +290, about +6 to about +280, about +6 to about +270, about +6 to about +260, about +6 to about +250, about +6 to about +240, about +6 to about +230, about +6 to about +220, about +6 to About +210, about +6 to about +200, about +6 to about +190, about +6 to about +180, about +6 to about +170, about +6 to about +160, about +6 to about +150, about +6 to about +140, about +6 to about +130 , About +6 to about +120, about +6 to about +110, about +6 to about +100, about +6 to about +90, about +6 to about +80, about +6 to about +70, about +6 to about +60, about +6 to about +50, about It is complementary to the targeted portion within the region of +6 to about +40, about +6 to about +30, or about +6 to about +20.

[0111] In some embodiments, the targeted portion of the LIPA SEC premRNA is a skippable exon 5'splice site (5'end) that can be skipped from -4e to a skippable exon 5'splice site (5'end). It is within the region up to + 2e with respect to the 3'end).

[0112] The ASO can be of any length suitable for effective enhancement of specific binding and splicing. In some embodiments, the ASO consists of 8-50 nucleobases. For example, the length of ASO is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, It can be 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases. In some embodiments, the ASO consists of more than 50 nucleobases. In some embodiments, the length of the ASO is 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20 nucleobases, 8 ~ 15 nucleic acid bases, 9-50 nucleic acid bases, 9-40 nucleic acid bases, 9-35 nucleic acid bases, 9-30 nucleic acid bases, 9-25 nucleic acid bases, 9-20 nucleic acid bases, 9-15 nucleic acid bases, 10-50 Nucleic acid bases, 10-40 nucleic acid bases, 10-35 nucleic acid bases, 10-30 nucleic acid bases, 10-25 nucleic acid bases, 10-20 nucleic acid bases, 10-15 nucleic acid bases, 11-50 nucleic acid bases, 11-40 nucleic acid bases. 11,35 nucleic acid bases, 11-30 nucleic acid bases, 11-25 nucleic acid bases, 11-20 nucleic acid bases, 11-15 nucleic acid bases, 12-50 nucleic acid bases, 12-40 nucleic acid bases, 12-35 nucleic acid bases, 12 ~ 30 nucleic acid bases, 12-25 nucleic acid bases, 12-20 nucleic acid bases, 12-15 nucleic acid bases, 13-50 nucleic acid bases, 13-40 nucleic acid bases, 13-35 nucleic acid bases, 13-30 nucleic acid bases, 13-25 Nucleic acid bases, 13 to 20 nucleic acid bases, 14 to 50 nucleic acid bases, 14 to 40 nucleic acid bases, 14 to 35 nucleic acid bases, 14 to 30 nucleic acid bases, 14 to 25 nucleic acid bases, 14 to 20 nucleic acid bases, 15 to 50 nucleic acid bases. 15, 40 nucleic acid bases, 15-35 nucleic acid bases, 15-30 nucleic acid bases, 15-25 nucleic acid bases, 15-20 nucleic acid bases, 20-50 nucleic acid bases, 20-40 nucleic acid bases, 20-35 nucleic acid bases, 20 ~ 30 nucleic acid bases, 20-25 nucleic acid bases, 25-50 nucleic acid bases, 25-40 nucleic acid bases, 25-35 nucleic acid bases, or 25-30 nucleic acid bases. In some embodiments, the ASO is 15 nucleotides in length. In some embodiments, the ASO is 16 nucleotides in length. In some embodiments, the ASO is 17 nucleotides in length. In some embodiments, the ASO is 18 nucleotides in length. In some embodiments, the ASO is 25 nucleotides in length.

[0113] In some embodiments, two or more ASOs with different chemistries but complementary to the same targeted portion of the SEC premRNA are used. In some embodiments, two or more ASOs that are complementary to different targeted portions of the SEC premRNA are used.

[0114] In some embodiments, the antisense oligonucleotides of the invention are one or more moieties or conjugates, such as targeted moieties or other conjugates that enhance the activity or intracellular uptake of the oligonucleotide. Chemically linked to. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholesteryl moieties, aliphatic chains such as dodecanediol groups or undecyl residues, polyamines or polyethylene glycol chains, or adamantane acetic acid. .. Oligonucleotides containing lipophilic moieties and methods of preparation are described in published literature. In certain embodiments, the antisense oligonucleotide is a debased nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (eg, mannose). Conjugates to moieties including, but not limited to, mannose-6-phosphate), lipids, or polyhydrogen compounds. Conjugates are understood in the art and, as described in the literature, for example, use linkers to place antisense oligonucleotides at several positions on sugar, base, or phosphate groups. It can be linked to any one or more nucleotides that contain. The linker may include a divalent or trivalent branch linker. In certain embodiments, the conjugate binds to the 3'end of the antisense oligonucleotide. Methods for preparing oligonucleotide conjugates are described, for example, in US Pat. No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," incorporated herein by reference.

[0115] In some embodiments, the nucleic acid targeted by ASO is the LIPA SEC pre-mRNA expressed intracellularly, such as in eukaryotic cells. In some embodiments, the term "cell" may refer to a population of cells. In some embodiments, the cells are within the subject. In some embodiments, cells are isolated from the subject. In some embodiments, the cells are ex vivo. In some embodiments, the cell is a cell or cell line associated with the condition or disease. In some embodiments, the cells are in vitro (eg, in cell culture).

Pharmaceutical composition
Pharmaceutical compositions or formulations that include the agents of the compositions described, eg, antisense oligonucleotides, for use in any of the described methods are well known to the pharmaceutical industry and are described in published literature. It can be prepared according to the conventional technique. In certain embodiments, the pharmaceutical composition or formulation for treating the subject is any antisense oligomer described herein, or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof. Includes an effective amount of. The pharmaceutical preparation containing the antisense oligomer can further contain a pharmaceutically acceptable excipient, diluent, or carrier.

[0117] Pharmaceutically acceptable salts are suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc. and have a reasonable benefit / risk factor. Is worth it. (See, for example, S.M. Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by this purpose). The salt can be prepared in situ during the final isolation and purification of the compound, or separately by reaction of the free base with a suitable organic acid. Examples of pharmaceutically acceptable non-toxic acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or acetic acid, oxalic acid, maleic acid, tartaric acid, citrate. A salt of an amino group formed with an organic acid such as an acid, succinic acid, or malonic acid, or by using other documented methods such as ion exchange. Other pharmaceutically acceptable salts include adipate, arginate, ascorbate, asparagate, benzenesulfonate, benzoate, bicarbonate, borate, butyrate, gypsum acid. Salt, Drosophila sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemi Sulfate, heptaneate, hexanate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malon Acid, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionic acid Salts, phosphates, picphosphates, pivalates, propionates, stearate, succinates, sulfates, tartrates, thiosocyanates, p-toluenesulfonates, undecanoates, valerate Etc. are included. Typical alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Additional pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, as well as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfones. Includes acid salts, and amine cations formed using counter ions such as aryl sulfonates.

[0118] In some embodiments, the composition is many possible, but not limited to, tablets, capsules, gel capsules, liquid syrups, softgels, suppositories, and enemas. Prescribed in dosage form. In some embodiments, the composition is formulated as a suspension in an aqueous, non-aqueous, or mixed medium. The aqueous suspension may further contain a substance that increases the consistency of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspending agent can also contain a stabilizer. In some embodiments, the pharmaceutical formulations or compositions of the invention include liquids, emulsions, microemulsions, foams, or liposome-containing formulations (eg, cationic or non-cationic liposomes). Not limited.

[0119] The pharmaceutical composition or formulation of the present invention, where appropriate, is one or more permeation enhancers, carriers, excipients, or other activities well known to those of skill in the art or described in the published literature. Alternatively, it can contain an inert ingredient. In some embodiments, liposomes also include sterically stabilized liposomes, such as liposomes containing one or more specified lipids. These specified lipids give rise to liposomes with enhanced circulating lifespan. In some embodiments, the sterically stabilized liposomes contain one or more glycolipids or are derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. In some embodiments, the surfactant is included in the pharmaceutical formulation or composition. The use of surfactants in pharmaceuticals, formulations, and emulsions is well known in the art. In some embodiments, the invention implements efficient delivery of antisense oligonucleotides with penetrating agents, eg, aids diffusion across cell membranes and / or enhances the permeability of lipophilic drugs. Let me. In some embodiments, the penetration enhancer is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating non-surfactant.

[0120] In some embodiments, the pharmaceutical formulation comprises a large number of antisense oligonucleotides. In some embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent.

Subject treatment
[0121] The compositions provided herein can be administered to an individual. The "individual" can be used interchangeably with the "subject" or "patient". The individual can be a mammal, such as a human or an animal such as a non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. In some embodiments, the individual is human. In some embodiments, the individual is a foetation, embryo, or child. In other embodiments, the individual can be another eukaryote, such as a plant. In some embodiments, the compositions provided herein are administered ex vivo to cells.

[0122] In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. In some embodiments, the individual has a genetic disorder such as any of the disorders described herein. In some embodiments, the individual is at risk of having a disease such as any of the diseases described herein. In some embodiments, the individual is at increased risk of having a disease or disorder caused by an inadequate amount of protein or inadequate activity of the protein. In some embodiments, the method involves prophylactic treatment if the individual is "increased risk" with a disease or disorder caused by an inadequate amount of protein or inadequate activity of the protein. For example, an individual may have an increased risk of having such a disease or disorder due to a family history of the disease. Typically, individuals at an increased risk of having such a disease or disorder will benefit from preventative measures (eg, by preventing or delaying the onset or progression of the disease or disorder). In some embodiments, the foetation is treated in the uterus, eg, by administering the ASO composition directly or indirectly (eg, via the mother) to the foetation.

[0123] The appropriate route of administration of ASO of the present invention may vary depending on the type of cell in which delivery of ASO is desirable. Many tissues and organs can be affected by CESD. In some embodiments, the liver may be the most significantly affected tissue. The ASO of the present invention can be administered parenterally to a patient by, for example, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

How to identify additional ASOs that prevent exon skipping
[0124] Methods for identifying or determining ASOs that prevent exon skipping of LIPA SEC premRNA are also within the scope of the present disclosure. For example, the method can include identifying or determining an ASO that prevents exon skipping of LIPA SEC premRNA. ASOs that specifically hybridize to different nucleotides within the target region of premRNA can be screened to identify or determine ASOs that improve the degree of skippable exon retention. In some embodiments, the ASO can block or interfere with the binding site of the splicing silencer. Any method known in the art is used to identify (determine) an ASO that produces the desired effect (eg, exon-containing, protein or functional RNA production) when hybridized to a skippable exon target region. )can do. These methods can also be used to identify ASOs that prevent exon skipping of skippable exons by binding to targeted regions within the intron adjacent to the skippable exons. Examples of possible methods are provided below.

[0125] ASO A screening round, referred to as a "walk," can be performed using an ASO designed to hybridize to the target region of premRNA. For example, the ASO used in the ASO walk can be targeted / skipped from approximately 100 nucleotides upstream of the 3'splice site of the skippable exon (eg, the sequence portion of the intron located upstream of the target / skippable exon). Approximately 100 nucleotides downstream of the 3'splice site of the exon and / or approximately 100 nucleotides downstream of the 5'splice site of the skippable exon to approximately 100 nucleotides downstream of the 5'splice site of the target / skippable exon ( For example, the sequence portion of the intron located downstream of the target / skippable exon) can be arranged every 5 nucleotides. For example, a first ASO of 18 nucleotides in length can be designed to specifically hybridize to +6 to +23 nucleotides to a target / skippable exon 5'splice site. The second ASO is designed to specifically hybridize to +11 to +28 nucleotides to the 5'splice site of the target / skippable exon. ASO is designed to span the target portion of premRNA. In some embodiments, the ASOs can be arranged closer together, eg, every 1, 2, 3, or 4 nucleotides. In addition, ASOs can be aligned from 100 nucleotides downstream of the 5'splice site to 100 nucleotides upstream of the 3'splice site. In some embodiments, the ASO can be aligned from about 572 nucleotides upstream of the 3'splice site to about 500 nucleotides downstream of the 5'splice site. In some embodiments, the ASO can be aligned from about 500 nucleotides upstream of the 3'splice site to about 572 nucleotides downstream of the 3'splice site.

[0126] One or more ASOs, or control ASOs (ASOs with scrambled sequences that are sequences that are not expected to hybridize to the target region), are, for example, target pre-mRNA (eg, SEC pre-mRNA described herein). Is delivered by hybridization to a disease-related cell line that expresses. Each exon-retaining effect of ASO is assessed by any method known in the art, eg, by reverse transcriptase (RT) -PCR using primers that span splice junctions, as described in the Examples. be able to. In ASO-treated cells, the increase or presence of longer RT-PCR products produced using primers spanning regions containing skippable exons is a skippable exon compared to control ASO-treated cells. It shows that the retention of is enhanced. In some embodiments, the exon retention efficiency, or the ratio of the retained skippable exon-containing mRNA to the exon-skipped mRNA, can be improved using the ASOs described herein. .. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine if each ASO achieved the desired effect (eg, enhanced functional protein production). Any method known in the art for assessing and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0127] ASO A second round of screening, referred to as a "microwalk," can be performed using an ASO designed to hybridize to the target region of premRNA. The ASOs used in the ASO Microwalk are ordered by nucleotide to further refine the nucleic acid sequence of the pre-mRNA that produces exon retention when hybridized with ASO.

[0128] Regions defined by ASOs that promote the inclusion of skippable exons in the target include ASOs spaced every nucleotide, as well as longer, typically 15-25 nucleotides ASOs. It will be explored in more detail by the ASO "Microwalk".

[0129] As described for ASO walks above, ASO microwalks include one or more ASOs, or control ASOs (ASOs with scrambled sequences that are sequences that are not expected to hybridize to the target region), eg. It is performed by delivery by hybridization to a disease-related cell line that expresses the target pre-mRNA. The splicing-inducing effect of each ASO uses any method known in the art, eg, as described herein (see eg, Examples), using a primer that spans skippable exons. It can be evaluated by reverse transcriptase (RT) -PCR. In ASO-treated cells, the increase or presence of longer RT-PCR products produced using primers spanning regions containing skippable exons is a skippable exon compared to control ASO-treated cells. It shows that the retention of is enhanced. In some embodiments, the exon retention efficiency, or the ratio of the retained skippable exon-containing mRNA to the exon-skipped mRNA, can be improved using the ASOs described herein. .. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine if each ASO achieved the desired effect (eg, enhanced functional protein production). Any method known in the art for assessing and / or quantifying protein production can be used, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

[0130] ASOs that produce exon-containing and increased protein production when hybridized to regions of premRNA are in vivo or diseased using animal models, such as transgenic mouse models in which the full-length human gene is knocked in. Can be tested in a humanized mouse model of. The appropriate route of administration of ASO may vary depending on the disease and / or cell type for which delivery of ASO is desirable. ASO can be administered, for example, by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. After administration, the cells, tissues, and / or organs of the model animal are evaluated, eg, splicing (efficiency, ratio, degree) and protein production by methods known in the art and described herein. By doing so, the effect of ASO treatment can be determined. The animal model can also be any phenotype or behavioral indication of the disease or severity of the disease.

[0131] The present invention will be described more specifically by the following examples. However, it should be understood that the present invention is by no means limited to these examples.
Example 1: Confirmation of exon skipping event in LIPA transcript by RT-PCR
[0132] RT-PCR was performed to reveal differences in the length of transcripts produced from the LIPA gene of fibroblasts obtained from healthy donors and donors with cholesteryl ester accumulation disease (CESD). Normal cells produced an mRNA transcript of the exon 8 carrying LIPA gene, yielding a full-length active LAL protein. When exon 8 is skipped, a mutation in the LIPA gene (c.894G> A) results in CESD and the non-functional LAL protein is predominantly expressed. Normal and CESD fibroblast mRNA transcripts were amplified using primers flanking exons 7 and 9 to detect the presence or absence of exons 8 in the produced LIPA transcripts. Polyacrylamide gel electrophoresis (PAGE) was then used to show the size of the resulting RT-PCR product. Control (normal) fibroblasts produce long PCR products containing exons 7, 8 and 9, whereas amplified PCR products from fibroblasts in CESD patients are short in length, exons 7 and 9 only. Consistent with the presence of what was retained in the transcript, exon 8 skipping was confirmed in fibroblasts of CESD patients. By this analysis, about 95% of the mRNA transcripts of CESD patients give rise to short-chain morphology (exon 8 is skipped), resulting in a non-functional LAL protein, and the full-length mRNA in which exon 8 is retained is It was shown to be produced about 5%. Product identity was confirmed by sequencing.
Example 2: Designing an ASO Walk Targeting Lipa Exxon 8
[0133] Using the methods described herein, the ASO Walk was designed to target exons 8 (Table 2, SEQ ID NO: 4) of LIPA (Table 1, SEQ ID NO: 1) (Table 3). , SEQ ID NOs: 54-63). Designed an ASO targeting skippable exons of LIPA SEC premRNA using regions immediately downstream of the 3'splice site of exon 8 and upstream of the 5'splice site of exon 8 extending from + 2e to -4e of nucleotides. bottom. Table 3 lists exemplary ASOs designed and their target sequences. From this design, 2'-MOE, PS backbone, 18-mer ASOs shifted at 5 nucleotide intervals are generated and utilized to target LIPA SEC pre-mRNA and increase LAL protein production.

Example 3: Designing an ASO Walk Targeting LIPA Intron 7
[0134] Using the methods described herein, the ASO Walk was designed to target Intron 7 (Table 2, SEQ ID NO: 3) of LIPA (Table 1, SEQ ID NO: 1) (Table 3). , SEQ ID NOs: 39-53). An ASO targeting intron 7 of the LIPA SEC premRNA was designed utilizing the region immediately upstream of the 3'splice site of intron 7 ranging from -16 to -100 of nucleotides. Table 3 lists exemplary ASOs designed and their target sequences. From this design, 2'-MOE, PS backbone, 18-mer ASOs shifted at 5 nucleotide intervals are generated and utilized to target LIPA SEC pre-mRNA and increase LAL protein production.
Example 4: Designing an ASO Walk Targeting LIPA Intron 8
[0135] Using the methods described herein, the ASO Walk was designed to target Intron 8 (FIG. 3, Table 2, SEQ ID NO: 2) of LIPA (Table 1, SEQ ID NO: 1). (Table 3, SEQ ID NOs: 1-38). An ASO targeting intron 8 of the LIPA SEC premRNA was designed utilizing the region immediately downstream of the 5'splice site of intron 8 spanning +6 to +100 of nucleotides. Table 3 lists exemplary ASOs designed and their target sequences. From this design, 2'-MOE, PS backbone, 18-mer ASOs shifted at 1 nucleotide intervals from +6 to +10 and then at 5 nucleotide intervals were created to target the LIPA SEC pre-mRNA to produce LAL protein. Was used to increase (see FIGS. 4A and 4B).
Example 5:
[0136] A series of experiments was designed to demonstrate the dose-dependent ability of ASOs targeting LIPA SEC pre-mRNA to alter transcription splicing to increase retention of intron 8 in mature mRNA products, which By c. Increased production of full-length functional LAL protein in donor CESD cells with 894G> A mutation. Cells are electroporated (eg, A-3334 (+9) in FIGS. 5, 7, and 9) or free uptake (eg, a set of concentrations of various 2'-MOE ASOs targeting intron 8). , A-3334 (+9) in FIG. 6), when the amount of ASO treated increased, the retention of exon 8 in the LIPA transcript produced in CESD fibroblasts was retained compared to untreated cells. It has been shown to result in an increase (eg, FIGS. 5A, 5C). A representative PAGE shows that the RT-PCR products for the two transcripts correspond to exon 8 content (overall, upper band) and exon 8 skipping (lower band), each quantified. It was evaluated as a percentage of the full-length transcript (FIGS. 5, 6 and 9). Additional analysis was performed to confirm that ASO treatment not only alters the production of mRNA transcripts, but also results in a dose-dependent increase in full-length functional LAL protein production in CESD cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) shows that the lysosomal acid lipase (LAL) protein is found in c. Fibroblasts of CESD patients with the 894G> A mutation were treated with increased amounts of 2'-MOE ASO targeting intron 8 at higher production levels compared to mock-treated cells (-). It shows that it increased (Fig. 7). The product corresponding to the glycosylated full-length protein was quantified and normalized to a Ponso S-stained blot. The activity of the LAL protein produced in ASO-treated CESD cells was confirmed using an enzyme assay. The results demonstrate that the expressed protein is functional and that the amount of activity is proportional to the amount of LAL protein (FIGS. 8 and 10).

[0137] Preferred embodiments of the present invention are set forth and described herein, but it will be apparent to those skilled in the art that such embodiments are provided merely by way of illustration. Numerous changes, changes, and replacements are here made by one of ordinary skill in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in the practice of the invention. It is intended that the following claims establish the scope of the present invention, and that the methods and structures within the scope of these claims, as well as their equivalents, are covered.

Claims (105)

A method of treating cholesteryl ester accumulation disease (CESD) in a subject requiring treatment by regulating the expression of target protein or functional RNA by the subject's cells, wherein the cells contain skippable exons. It has pre-mRNA (SEC pre-mRNA), and the SEC pre-mRNA is added to the skippable exson, the intron adjacent to the 5'splice site of the skippable exson, and the 3'splice site of the skippable exson. Containing an adjacent intron, the SEC premRNA encodes the target protein or functional RNA, the method comprises targeting the target cell with the target portion of the SEC premRNA encoding the target protein or functional RNA. Contact with an antisense oligomer (ASO) complementary to, thereby retaining the skippable exson in the mRNA processed from the SEC premRNA encoding the target protein or functional RNA, thereby. The method comprising regulating the level of mRNA encoding the target protein or functional RNA and regulating the expression of the target protein or functional RNA in the target cell. A method of regulating the expression of a target protein, lysosomal acid lipase (LAL), by cells having a skippable exon-containing premRNA (SEC premRNA), wherein the SEC premRNA is the skippable exon, said. The SEC premRNA encodes the LAL protein, which comprises an intron flanking the 5'splice site of the skippable exson and an intron flanking the 3'splice site of the skippable exson. Contact with an antisense oligomer (ASO) complementary to the targeted portion of the SEC premRNA encoding the LAL protein, whereby the skippable exson is processed from the SEC premRNA encoding the LAL protein. The method comprising regulating the level of mRNA encoding the LAL protein and, thereby regulating the expression of the LAL protein, in said mRNA. The method of claim 1, wherein regulating the expression or level of the target protein or mRNA encoding the target protein increases the expression or level of the target protein or mRNA encoding the target protein. The method of claim 2, wherein regulating the expression or level of the LAL protein or mRNA encoding the LAL protein increases the expression or level of the LAL protein or mRNA encoding the LAL protein. The method according to claim 1 or 3, wherein the target protein is LAL. The method according to any one of claims 1 to 5, wherein the cells are in or derived from a subject having a pathology caused by a deficiency in the amount or activity of the LAL protein. Claimed that at least one of the nine nucleotides at +1 to +6 of the intron adjacent to the 5'splice site and -3e to -1e of the skippable exon contains at least one mutation. Item 6. The method according to any one of Items 1 to 6. The method of claim 7, wherein the mutation is a substitution. The method of claim 7, wherein the mutation is in -1e. The mutation is c. The method according to claim 7, wherein 894G> A. The method according to any one of claims 1 to 10, wherein the deficiency in the amount of the target protein is caused by autosomal recessive inheritance. The subject has a first allele encoding a functional target protein and (a) a second allele encoding a functional target protein, and the antisense oligomer is the first or second allele. It binds to the targeted portion of the SEC premRNA transcribed from the gene or has the first allele and (b) the second allele, and the antisense oligomer is from the first allele. 11. The method of claim 11, which binds to the targeted portion of the transcribed SEC premRNA. The subject has a condition caused by a disorder caused by a deficiency in the amount or function of the target protein.
(A) A first allele containing the first mutation, which results from the first mutation.
(I) Whether the target protein is produced at a reduced level compared to production from a wild-type allele.
(Ii) The target protein is produced in a form having reduced functionality compared to an equivalent wild-type protein, or (iii) the target protein is not produced.
A first allele and (b) a second allele containing a second mutation, due to the second mutation.
(I) Whether the target protein is produced at a reduced level compared to production from a wild-type allele.
(Ii) The target protein is produced in a form having reduced functionality compared to an equivalent wild-type protein, or (iii) the target protein is not produced.
Second allele,
The method according to any one of claims 1 to 12, wherein the first and second mutations are the same or different. 13. The method of claim 13, wherein the target protein is produced in a form having reduced functionality compared to an equivalent wild-type protein. 13. The method of claim 13, wherein the target protein is produced in a fully functional form as compared to an equivalent wild-type protein. The targeted portion of the SEC premRNA ranges from -4e to the 5'splice site of the skippable exon within the skippable exon to + 2e to the 3'splice site of the skippable exon. The method according to any one of claims 1 to 15, which is within the region. The targeted portion of the SEC premRNA is
(A) within the +6 to +500 region of the intron adjacent to the skippable exon 5'splice site, or (b) the -16 to -500 region of the intron adjacent to the skippable exon 3'splice site. Inside,
The method according to any one of claims 1 to 15. The targeted portion of the SEC premRNA is
(A) Within the region of -4e to -500e with respect to the skippable exon 5'splice site,
(B) Within the region +6 to +500 with respect to the skippable exon 5'splice site,
(C) Within the region -16 to -500 with respect to the skippable exon 3'splice site, or (d) within the region + 2e to + 500e with respect to the skippable exon 3'splice site.
The method according to any one of claims 1 to 15. Claimed that the SEC premRNA comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity in any one of SEQ ID NO: 1 or its complementary sequence. Item 2. The method according to any one of Items 1 to 18. Claim 1 the SEC premRNA is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 1 or its complementary sequence. The method according to any one of 18 to 18. The targeted portion of the SEC premRNA is at least 80%, 85%, 90%, 95%, 97%, or in a region containing at least 8 contiguous nucleic acids of SEQ ID NOs: 2-4 or their complementary sequences. The method of any one of claims 1-20, comprising a sequence having 100% sequence identity. Claimed that the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% identity in any one of SEQ ID NOs: 5-63 or their complementary sequences. Item 2. The method according to any one of Items 1 to 21. The targeted portion of the SEC premRNA is within the region from -4e to the 5'splice site of the skippable exon to + 2e to the 3'splice site of the skippable exon and said skip. The method according to any one of claims 1 to 22, wherein the possible exon is exon 8. Claimed that the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% identity in any one of SEQ ID NOs: 54-63 or their complementary sequences. Item 23. 15. The method according to any one item. Claimed that the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% identity in any one of SEQ ID NOs: 5-38 or their complementary sequences. Item 25. Claims 1-that the targeted portion of the SEC pre-mRNA is in the region -16 to -500 with respect to the 3'splice site of the skippable exon and the skippable exon is exon 8. The method according to any one of 22. Claimed that the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% identity in any one of SEQ ID NOs: 39-53 or their complementary sequences. Item 27. 13. The method described. The method according to any one of claims 1 to 29, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA. The method according to any one of claims 1 to 30, wherein the target protein produced is a full-length protein, a wild-type protein, or a combination thereof. The total amount of the mRNA encoding the target protein or functional RNA produced in the cell in contact with the antisense oligomer is the mRNA encoding the target protein or functional RNA produced in the control cell. About 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 compared to the total amount of ~ About 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 ~ About 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to About 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times The method according to any one of claims 1-31, which increases at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times. The total amount of the mRNA encoding the target protein or functional RNA produced in the cell in contact with the antisense oligomer is the mRNA encoding the target protein or functional RNA produced in the control cell. About 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20%. ~ About 100%, about 20% ~ about 150%, about 20% ~ about 200%, about 20% ~ about 250%, about 50% ~ about 100%, about 50% ~ about 150%, about 50% ~ about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250% , About 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% increase The method according to any one of claims 1 to 31. The total amount of target protein produced by the cells in contact with the antisense oligomer is about 1.1 to about 10 times, about 1.5 to about 1.5 to about, compared to the total amount of target protein produced by the control cells. 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times , About 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to About 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times , Or the method according to any one of claims 1-33, which increases by at least about 10 times. The total amount of target protein produced by the cells in contact with the antisense oligomer is about 20% to about 300%, about 50% to about 300%, as compared to the total amount of target protein produced by the control cells. , About 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% ~ About 200%, about 100% ~ about 250%, about 150% ~ about 200%, about 150% ~ about 250%, about 200% ~ about 250%, at least about 10%, at least about 20%, at least about 50 %, At least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, the method of any one of claims 1-33. The method of any one of claims 1-35, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. Any of claims 1-36, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-fluoro, or a 2'-O-methoxyethyl moiety. The method according to item 1. The method according to any one of claims 1 to 37, wherein the antisense oligomer contains at least one modified sugar moiety. 38. The method of claim 38, wherein each sugar moiety is a modified sugar moiety. The antisense oligomer contains 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, and 8 to 20 nucleobases. Nucleic Acid Bases, 8 to 15 Nucleic Acid Bases, 9 to 50 Nucleic Acid Bases, 9 to 40 Nucleic Acid Bases, 9 to 35 Nucleic Acid Bases, 9 to 30 Nucleic Acid Bases, 9 to 25 Nucleic Acids Bases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 11 30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases 1 to any one of claims 1 to 39, which comprises 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. The method described. The antisense oligomer complements the targeted portion of the SEC premRNA encoding the protein by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. The method according to any one of claims 1 to 40, which has the property. The method of any one of claims 1-41, further comprising assessing LIPA protein expression. Claim that CESD is treated and the antisense oligomer binds to a targeted portion of the LIPA SEC premRNA, wherein the targeted portion comprises at least 8 contiguous nucleic acids of SEQ ID NOs: 2-4 or their complementary sequences. The method according to any one of 1-42. The method according to any one of claims 1 to 43, wherein the subject is a human. The method according to any one of claims 1 to 44, wherein the subject is a non-human animal. The method according to any one of claims 1 to 45, wherein the subject is a foetation, an embryo, or a child. The method according to any one of claims 1 to 46, wherein the cells are ex vivo contacted. The antisense oligomer is administered by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection into the subject, according to any one of claims 1 to 47. The method described. The 16 nucleotides at -15 to -1 of the intron and + 1e of the skippable exon adjacent to the 3'splice site are identical to the corresponding wild-type sequence, and the intron is intron 7. The method according to any one of claims 1 to 48, wherein the skippable exon is an exon 8. The antisense oligomer used in the method according to any one of claims 1-49. An antisense oligomer comprising a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity in any one of SEQ ID NOs: 5-63 or their complementary sequences. A pharmaceutical composition comprising the antisense oligomer according to claim 50 or 51 and a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition according to claim 52 is administered by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection to treat a subject in need of treatment. Method. To be used in a method of regulating the expression of target protein or functional RNA by cells for treating cholesteryl ester accumulation disease (CESD) associated with deficient protein or deficient functional RNA in subjects in need of treatment. The deficient protein or deficient functional RNA is deficient in amount or activity in the subject, and the antisense oligomer encodes the target protein or the functional RNA. Reduces the removal of exons from the skippable exon-containing premRNA (SEC premRNA), the target protein is the missing protein, the functional RNA is the missing RNA, and the SEC premRNA is said. The skippable exson comprises an intron adjacent to a skippable exson, an intron adjacent to the 5'splice site of the skippable exson, and an intron adjacent to the 3'splice site of the skippable exson, wherein the skippable exson is the target protein or said. The composition, which is retained in an mRNA processed from the SEC pre-mRNA encoding a functional RNA, thereby regulating the production or activity of the target protein or the functional RNA in the subject. A composition comprising an antisense oligomer for use in a method of treating a pathological condition associated with a lysosomal acid lipase (LAL) protein in a subject in need of treatment, wherein the method is the LAL protein by the subject's cells. The cell comprises a skippable exon, an intron flanking the 5'splice site of the skippable exon, and an intron flanking the 3'splice site of the skippable exon. It has a skippable exon-containing premRNA (SEC premRNA), wherein the SEC premRNA encodes the LAL protein, the method of contacting the cell with the antisense oligomer, thereby the skippable. Exons are retained in the mRNA processed from the SEC pre-mRNA transcript encoding the LAL protein, thereby regulating the level of the mRNA encoding the LAL protein and expressing the LAL protein in said cells of interest. The composition comprising adjusting for. Modulating the activity, expression, and / or production of the target protein or mRNA encoding the target protein increases the activity, expression, and / or production of the target protein or mRNA encoding the target protein. The composition according to claim 54. Claim 54, wherein regulating the activity, expression, and / or production of the LAL protein or mRNA encoding the LAL protein increases the activity, expression, and / or production of the LAL protein or mRNA encoding the LAL protein. The composition described. The composition according to claim 54 or 56, wherein the target protein is LAL. The composition according to claim 55, wherein the condition is a disease or disorder. The composition of claim 59, wherein the disease or disorder is CESD. The composition according to claim 60, wherein the target protein and SEC pre-mRNA are encoded by the LIPA gene. The SEC pre is in the region from -4e to the 5'splice site of the skippable exon in the skippable exon to + 2e to the 3'splice site of the skippable exon. The composition according to any one of claims 54 to 61, which targets a portion of mRNA. The antisense oligomer is in the skippable exon.
(A) within the region +6 to +500 with respect to the skippable exon 5'splice site, or (b) within the region -16 to -500 with respect to the skippable exon 3'splice site.
The composition according to any one of claims 54 to 61, which targets a portion of the SEC premRNA in the above. The portion of the SEC premRNA in which the antisense oligomer is located within a region approximately 100 nucleotides downstream of the 5'splice site of the skippable exon to approximately 100 nucleotides upstream of the 3'splice site of the skippable exon. The composition according to any one of claims 54 to 61, which is targeted. The targeted portion of the SEC premRNA is
(A) within the +6 to +500 region of the intron adjacent to the 5'splice site of the skippable exon, or (b) -16 to -500 of the exon adjacent to the 3'splice site of the skippable exon. In the area,
The composition according to any one of claims 54 to 61. The targeted portion of the SEC premRNA is
(A) Within the region of -4e to -500e with respect to the skippable exon 5'splice site,
(B) Within the region +6 to +500 with respect to the skippable exon 5'splice site,
(C) Within the region -16 to -500 with respect to the skippable exon 3'splice site, or (d) within the region + 2e to + 500e with respect to the skippable exon 3'splice site. , The composition according to any one of claims 54 to 61. The composition according to any one of claims 54 to 61, wherein the target protein is LAL. Claimed that the SEC premRNA comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity in any one of SEQ ID NO: 1 or its complementary sequence. Item 67. Claim 67, wherein the SEC premRNA is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 1 or its complementary sequence. The composition according to. The targeted portion of the SEC premRNA is at least 80%, 85%, 90%, 95%, 97%, or in a region containing at least 8 contiguous nucleic acids of SEQ ID NOs: 2-4 or their complementary sequences. The composition of claim 67, comprising a sequence having 100% sequence identity. Claimed that the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% identity in any one of SEQ ID NOs: 5-63 or their complementary sequences. Item 67. The targeted portion of the SEC premRNA is within the region from -4e to the 5'splice site of the skippable exon to + 2e to the 3'splice site of the skippable exon and said skip. 67. The composition of claim 67, wherein the possible exons are exons 8. Claimed that the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% identity in any one of SEQ ID NOs: 54-63 or their complementary sequences. Item 72. 67. The target portion of the SEC premRNA is in the region -16 to -500 with respect to the 3'splice site of the skippable exon, and the skippable exon is exon 8. The composition described. Claimed that the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% identity in any one of SEQ ID NOs: 5-38 or their complementary sequences. Item 72. 67. Composition. Claimed that the ASO comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% identity in any one of SEQ ID NOs: 39-53 or their complementary sequences. Item 72. The composition according to any one of claims 54 to 77, wherein the SEC pre-mRNA is produced by partial splicing of a full-length pre-mRNA or a wild-type pre-mRNA. The composition according to any one of claims 54 to 78, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA. The composition according to any one of claims 54 to 79, wherein the target protein produced is a full-length protein or a wild-type protein. The composition according to any one of claims 54 to 80, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. The composition according to any one of claims 54 to 81, wherein the antisense oligomer is an antisense oligonucleotide. Any of claims 54-82, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-fluoro, or a 2'-O-methoxyethyl moiety. The composition according to item 1. The composition according to any one of claims 54 to 83, wherein the antisense oligomer contains at least one modified sugar moiety. The composition according to claim 84, wherein each sugar moiety is a modified sugar moiety. The antisense oligomer contains 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, and 8 to 20 nucleobases. Nucleic Acid Bases, 8 to 15 Nucleic Acid Bases, 9 to 50 Nucleic Acid Bases, 9 to 40 Nucleic Acid Bases, 9 to 35 Nucleic Acid Bases, 9 to 30 Nucleic Acid Bases, 9 to 25 Nucleic Acids Bases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 11 30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases Nucleic acid bases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases according to any one of claims 54 to 85. The composition described. A pharmaceutical composition comprising an antisense oligomer of any of the compositions according to claims 54-86 and a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition according to claim 87 is administered by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection to treat a subject in need of treatment. Method. A pharmaceutical composition comprising an antisense oligomer that hybridizes to the target sequence of a LIPA mRNA transcript and a pharmaceutically acceptable excipient, diluent, or carrier, wherein the LIPA mRNA transcript is skipped. The pharmaceutical composition comprising exons, wherein the antisense oligomer induces retention of exons skipped from the LIPA mRNA transcript. The pharmaceutical composition according to claim 89, wherein the LIPA mRNA transcript is a LIPA SEC pre-mRNA transcript. The targeted portion of the LIPA SEC pre-mRNA transcript is relative to the 5'splice site of the skippable exon within the skippable exon from -4e to the 3'splice site of the skippable exon. The pharmaceutical composition according to claim 89 or 90, which is within the region up to + 2e. The LIPA SEC pre-mRNA transcript is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 in any one of SEQ ID NO: 1 or its complementary sequences. The pharmaceutical composition according to claim 89 or 90, which is encoded by a gene sequence having% sequence identity. The LIPA SEC pre-mRNA transcript is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 in any one of SEQ ID NO: 1 or its complementary sequences. The pharmaceutical composition according to claim 89 or 90, comprising a sequence having% sequence identity. The pharmaceutical composition according to claim 89, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. The pharmaceutical composition according to claim 89, wherein the antisense oligomer is an antisense oligonucleotide. The pharmaceutical composition according to claim 89, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-fluoro, or a 2'-O-methoxyethyl moiety. thing. The pharmaceutical composition according to claim 89, wherein the antisense oligomer contains at least one modified sugar moiety. The antisense oligomer contains 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, and 8 to 20 nucleobases. Nucleic Acid Bases, 8 to 15 Nucleic Acid Bases, 9 to 50 Nucleic Acid Bases, 9 to 40 Nucleic Acid Bases, 9 to 35 Nucleic Acid Bases, 9 to 30 Nucleic Acid Bases, 9 to 25 Nucleic Acids Bases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 11 30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases 89. The pharmaceutical composition according to claim 89, which comprises 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. The antisense oligomer has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementarity to the targeted portion of the LIPA SEC pre-mRNA transcript. The pharmaceutical composition according to claim 89 or 90. The pharmaceutical composition according to claim 89 or 90, wherein the targeted portion of the LIPA SEC pre-mRNA transcript is in a sequence selected from SEQ ID NOs: 2-4 or complementary sequences thereof. The antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% in any one of SEQ ID NOs: 5-63 or their complementary sequences. 89. The pharmaceutical composition of claim 89, comprising a nucleotide sequence having 97%, 98%, or 99% sequence identity. The pharmaceutical composition according to claim 89, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 5-63 or a complementary sequence thereof. The pharmaceutical composition according to any one of claims 89 to 102, which is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. A method of treating a subject having a pathology caused by a deficiency in the amount or activity of the LAL protein, at least about 80%, 85%, 90% of any one of SEQ ID NOs: 5-63 or their complementary sequences. Included in administering to said subject an antisense oligomer comprising a nucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. The method. The method of claim 104, wherein the subject is a human.

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